Structure-activity studies in the development of a hydrazone based inhibitor of adipose-triglyceride lipase (ATGL)

Article abstract of DOI:10.1016/j.bmc.2015.02.051

Adipose triglyceride lipase (ATGL) catalyzes the degradation of cellular triacylglycerol stores and strongly determines the concentration of circulating fatty acids (FAs). High serum FA levels are causally linked to the development of insulin resistance a

Full text of DOI:10.1016/j.bmc.2015.02.051

Bioorganic & Medicinal Chemistry 23 (2015) 2904–2916
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity studies in the development of a hydrazone based
inhibitor of adipose-triglyceride lipase (ATGL)
Nicole Mayer a, , Martina Schweiger b, , Michaela-Christina Melcher a, Christian Fledelius c,
Rudolf Zechner b, Robert Zimmermann b, , Rolf Breinbauer a,
⇑
⇑
a Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria
b Institute of Molecular Biosciences, University of Graz, Heinrichstraße 31/II, A-8010 Graz, Austria
c Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Adipose triglyceride lipase (ATGL) catalyzes the degradation of cellular triacylglycerol stores and strongly
determines the concentration of circulating fatty acids (FAs). High serum FA levels are causally linked to
the development of insulin resistance and impaired glucose tolerance, which eventually progresses to
overt type 2 diabetes. ATGL-specific inhibitors could be used to lower circulating FAs, which can counter-
act the development of insulin resistance. In this article, we report about structure–activity relationship
(SAR) studies of small molecule inhibitors of ATGL based on a hydrazone chemotype. The SAR indicated
that the binding pocket of ATGL requests rather linear compounds without bulky substituents. The best
Received 29 January 2015
Revised 24 February 2015
Accepted 25 February 2015
Available online 5 March 2015
Keywords:
Adipose triglyceride lipase
Hydrazones
Inhibitor
Lipid metabolism
Triglycerides
inhibitor showed an IC50 = 10 lM in an assay with COS7-cell lysate overexpressing murine ATGL.
Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
1. Introduction
data demonstrate that ATGL-deficient mice exhibit low plasma
FA levels, improved glucose tolerance and insulin sensitivity, and
During the last decades increasing evidence has emerged that
fatty acid (FA) metabolism is closely linked to the development
of metabolic disorders. Increased circulating FAs, as observed in
obesity, can cause FA overload of non-adipose tissues resulting in
ectopic triglyceride (TG) accumulation which is associated with
impaired metabolic functions of these tissues, insulin resistance,
and inflammation.1 Thus, decreasing plasma FA levels represents
an apparent strategy to counteract the development of metabolic
disease.
Plasma FA levels are strongly determined by lipases catalyzing
the degradation of TG stores in adipose tissue. Efficient FA
mobilization requires the activity of two lipases: Adipose triglyc-
eride lipase (ATGL) and hormone-sensitive lipase (HSL). ATGL
releases the first FA from the TG molecule and generates diacyl-
glycerol (DG).2 Hormone-sensitive lipase (HSL) is the rate-limiting
enzyme for the hydrolysis of DG,3 but is also capable of hydrolyz-
ing TG and monoglycerides.4,5 Since ATGL performs the first and
rate-limiting step in lipolysis, inhibition of this enzyme is a
powerful way to reduce FA availability. Accordingly, published
are resistant to high-fat diet-induced insulin resistance.6–9
Furthermore, pharmacological inhibition of ATGL reduces circulat-
ing FAs and triglyceride levels.10
It is important to note that defective ATGL function is associated
with systemic TG accumulation in humans and rodents.6 Humans
with defective ATGL function develop cardiomyopathy at the age
of ꢀ30 years due to massive accumulation of TG in cardiomy-
ocytes.11 Thus, it is reasonable to assume that also inhibitor-medi-
ated ablation of ATGL activity can cause cardiac TG accumulation
and myopathy. However, transient or partial inhibition of ATGL
must not necessarily result in cardiac TG accumulation, since tis-
sue TG stores can be rapidly mobilized to generate FA for energy
conversion or as building blocks for complex lipids. Furthermore,
small molecule inhibitors often show specific tissue distribution
patterns which avoid their accumulation in cardiomyocytes, as
recently demonstrated for the ATGL inhibitor Atglistatin.10
The significant biological relevance of ATGL makes it desirable
to identify inhibitors of ATGL as tool compounds to study its bio-
logical role and validate ATGL as
a potential drug target.
However, at present no 3D-structure of ATGL is available and any
effort to identify inhibitors of ATGL has to rely on the traditional
approach of synthesizing and testing compounds. Recently, we
have described a first inhibitor of murine ATGL, Atglistatin (B),10
⇑
Corresponding authors. Fax: +43 316 873 32400.
E-mail addresses: robert.zimmermann@uni-graz.at (R. Zimmermann), brein-
bauer@tugraz.at (R. Breinbauer).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2015.02.051
0968-0896/Ó 2015 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

N. Mayer et al. / Bioorg. Med. Chem. 23 (2015) 2904–2916
2905
which has been developed from a biaryl hit compound A resulting
from high-throughput screening (HTS) campaign originally
aimed to inhibit HSL. In parallel to that work we have also followed
a second hit compound 1a based on a hydrazone chemotype,
which had slightly better initial activity against ATGL
benzaldehydes in toluene at 100 °C producing the hydrazones
1a–1z in 44–99% yield (Table 1).15 Similarly, another set of ten ali-
phatic and aromatic aldehydes was reacted with the same hydra-
zine under similar conditions to produce the hydrazones 2a–2j in
82–99% yield (Scheme 2, Table 2).
a
(IC50 = 110
l
M). Here we report about our efforts to develop 1a
In the second stage of our studies we put our focus on the
piperazine function by exchanging on the one hand the N-phenyl
substituent at the piperazine by other N-aryl and N-alkyl sub-
stituents (compounds 3a–3g, Table 3) and on the other hand by
replacing the piperazine with a piperidine or homopiperazine ring
(compounds 3h–3j, Table 3). The proven substitution pattern from
the hit compound representing the 2,4-dihydroxyphenylic system
was kept constant in the aldehyde reactant in these studies
(Scheme 3).
as a tool compound and discuss the structure–activity data gained
from this hydrazone chemotype (Fig. 1).
2. Results and discussion
2.1. Chemistry
Lead structure 1a resulting from the HTS can be characterized
by a hydrophobic side chain represented by the phenylpiperazine
moiety connected with a catechol moiety via a hydrazone linkage.
While we were aware about the problem associated with the
intrinsic toxicity associated with hydrazones, we found the ready
accessibility and synthetic flexibility of hydrazones attractive to
gain fast insight into the structure–activity relationship of this
inhibitor chemotype and hoped to be able to replace the hydrazone
later by a more innocent functional group.12
The synthesis of the homopiperazine compound 3j started with
a Pd(0)-catalyzed Buchwald–Hartwig-arylation of Boc-protected
homopiperazine (4) with bromobenzene using Pd(OAc)2/X-Phos
with NaOtBu as base,16 which after deprotection with HCl deliv-
ered the homopiperazine as its hydrochloride salt 5 in moderate
29% yield (Scheme 4). Nitrosylation of 5 and reduction with
14
LiAlH4 produced hydrazine 6 in 24% yield, which could then
smoothly react with 2,4-dihydroxybenzaldehyde to the desired
condensation product 3j in 97% yield.
In the first stage of our optimization studies we synthesized a
set of compounds 1b–1z, in which we varied the catechol sub-
stituent of hit compound 1a by differently substituted aryl and ali-
phatic aldehydes in the hydrazone forming process (Scheme 1).
Starting from commercial N-phenylpiperazine nitrosation with
In the final stage of our structure activity studies we attempted
to replace the hydrazone linkage with other linking units
(Scheme 5). The more stable, but by one atom shorter, test com-
pound 8 was prepared by reductive amination of 3,4-dihydroxyben-
zaldehyde (7) and N-phenylpiperidine with NaBH(OAc)3.17 For
reasons of synthetic accessibility the 2,4-dihydroxyphenylic system
was replaced in the synthesis of the following test compounds by
other successful aldehyde fragments from the first stage of
optimization, which did not have the reactive phenolic OH
groups and were replaced by MeO and F substituents, which
would also be less prone to phase 2 metabolism.12 The hydrazide
analog 10 was synthesized via reaction of the acid chloride
tert-butylnitrite13 followed by reduction with LiAlH4 delivered
14
the desired 4-phenylpiperazin-1-amine in 74% yield. This central
nucleophile was then reacted with 26 differently substituted
HTS-hits
H
N
O
N
N
HO
O
prepared from
9
with 4-phenylpiperazin-1-amine in 53%
O
overall yield. For the synthesis of urea analog 12, aniline 11 was
first converted into the Boc-carbamate, which was then reacted
with N-lithiated phenylpiperazine delivering 12 in 25% overall
yield.18 Finally, urethane 14 was prepared by following a strategy,19
in which phenol 13 is first activated with 1,10-carbonyldiimidazole
(CDI) to an aryloxycarbonylimidazol and then reacted with
1-phenylpiperazine to produce the desired carbamate in 20% overall
yield.
N
N
N
Atglistatin
IC50 = 120 µM
B
A
HO
HO
this work
2.2. Biological activity
N
N
N
IC50 = 110 µM
All synthesized test compounds were screened for their effi-
ciency to inhibit ATGL activity in vitro in an assay using radiola-
belled triolein as substrate.20 As ATGL cannot be purified, the
assays were performed with COS7-cell lysates from cells
1a
Figure 1. Hit compounds which served for the development of tool compounds
against ATGL.
O
R4
R3
H
R1
R2
1) 1.55 eq tBuONO
toluene, 100 °C
R4
THF, reflux
H2N
N
N
HN
N
2) 2.2 eq LiAlH4
THF, reflux, 3 h
74 %
R3
N
N
N
R2
R1
1a-1z
Scheme 1. Synthesis of test compounds via hydrazone formation with aliphatic and arylic aldehydes.

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N. Mayer et al. / Bioorg. Med. Chem. 23 (2015) 2904–2916
Table 1
Screening data of hydrazones 1a–z produced via Scheme 1
Entry
Compound
R1
R2
R3
R4
Yield (%)
IC50 (%)
I200 (%)
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
–H
–H
–H
–H
–H
–OH
–H
–OH
–H
–H
–OH
–OMe
–OH
–OMe
–OH
–H
–OH
–H
–OnPr
–OPh
–H
–OMe
–F
–Cl
–H
–H
–H
–H
–H
–H
–H
–H
–H
–OH
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–OMe
–OMe
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
92
99
99
68
50
99
99
99
71
94
83
44
90
90
95
97
89
99
92
99
61
96
84
87
95
99
110
100
80
60
70
75
50
10
>200
>200
>200
>200
75
>200
>200
180
>150
120
50
>200
>200
>200
>200
>200
>200
>200
69
65
56
82
71
64
85
94
14
24
43
41
67
20
42
60
73
60
76
22
5
–OMe
–OMe
–OH
–H
–H
–H
–OH
–H
–H
–OMe
–OMe
–F
–Cl
–Cl
–H
–H
–H
–CN
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
–OMe
–H
–H
–H
–Cl
–NO2
–Br
–CN
–H
–H
–H
–H
–H
1s
1t
–CH3
–NMe2
–NHAc
–N(Me)(CH2)2OH
–OCH2O–
–O(CH2)2O–
–NHCONH–
1u
1v
1w
1x
1y
1z
14
5
64
27
16
–H
–H
–H
–OCH2O–
–O(CH2)2O–
–NHCONH–
Table 3
O
R
R
toluene, 100 °C
+
H2N
N
N
N
N
N
Screening data of hydrazones 3a–j produced via Scheme 3
H
Entry Compound Yield (%) IC50 (%) I200 (%)
R
2a-j
1
2
3a
3b
N
N
N
N
N
74
25
>200
50
19
78
Scheme 2. Synthesis of test compounds via hydrazone formation with aliphatic
and arylic aldehydes.
F
Table 2
3
4
5
3c
3d
3e
58
95
95
>200
>200
>200
44
48
14
N
N
N
N
Screening data of hydrazones 2a–j produced via Scheme 2
Entry
Compound
R
Yield (%)
IC50 (%)
I200 (%)
1
2
2a
2b
94
99
>200
>200
12
12
3
4
2c
98
94
200
130
49
58
N
N
N
N
N
N
N
2d
6
7
8
3f
66
76
57
>200
>200
40
25
36
81
3g
3h
5
2e
87
>200
16
N
6
7
2f
99
99
>200
140
46
60
N
N
2g
9
3i
3j
95
97
>200
>200
26
87
N
N
8
9
2h
2i
82
83
>200
>200
<1
8
10
N
N
10
2j
98
200
49
IC50-values and the inhibition of the enzyme using an inhibitor
concentration of 200 M are listed. Starting from the hit compound
l
1a from the HTS (entry 1) the influence of the HO substituents on
the biological activity was investigated. As catechols are known to
be oxidation sensitive and to be metabolized very fast in organ-
isms, we varied the amount and position of HO substituents (1e–
1h) and partly replaced them by methoxy groups (1b–1d). The
activity increased in comparison to 1a if one of the HO groups
was methylated (entries 3 and 4). Even more pronounced was
the activity gain if only a single HO substituent was present at
overexpressing recombinant murine ATGL. For the determination
of the IC50-value inhibitors were administered with increasing con-
centrations to the enzyme and its substrate. To determine the
ATGL-inhibition efficiency at 200 lM (I200), 200 lM inhibitor was
administered to the enzyme and its substrate. The results of the
first set of test compounds 1a–1z are summarized in Table 1. The

N. Mayer et al. / Bioorg. Med. Chem. 23 (2015) 2904–2916
2907
substituents or multiple substitution leading to sterically demand-
ing compounds resulted in inactive compounds (entries 9–12 and
20–26). Interestingly, the rather linear 4-cyanosubstituted com-
O
H
toluene, 100 °C
HO
H2N
R
HO
+
N
R
OH
OH
pound 1s (IC50 = 50 lM, entry 19) had encouraging activity, while
3a-j
the bulkier 4-methylsubstituted analog 1t (entry 20) was inactive,
providing further evidence for a rather small and linear binding
pocket of ATGL.
Scheme 3. Synthetic access to second set of test compounds by variation of the
piperazine moiety.
In the second set of screening compounds, the phenyl group of
the aldehyde was replaced by alkyl, aryl and hetaryl groups
(Table 2). From this set of compounds only the 1-cyclohexen-1-yl
compound 2d (entry 4) and 4-pyridyl compound 2g (entry 7)
showed moderate activity, while all other compounds were more
or less inactive, corroborating the results from Table 1 that struc-
tures bulkier than the phenyl ring in hit compound 1a seem to
fit less well into the binding pocket.
the aldehyde building block (entries 5–7). The highest activity was
observed for compound 1h with its 2,4-dihydroxyphenyl moiety
(IC50 = 10
1a were substituted by fluorines the activity was slightly improved
(IC50 = 75 M, 1m, entry 13), whereas the corresponding Cl-analog
lM, entry 8). If the HO substituents in the hit compound
l
1n was inactive (entry 14) as also its structural isomer (entry 15).
The comparison of ortho-hydroxylated compound 1f with the
analogous substrates 1p–1r showed that –NO2, –Br or –CN sub-
stitution led to less active compounds (entries 16–18). From the
data of the first screening it also became apparent, that bulky
In the next stage of our optimization studies we kept the best
scoring fragment 2,4-dihydroxyphenyl (identified above from the
screening of compounds in Tables 1 and 2) and varied the N-
1) 5% Pd(OAc)2
5 X-Phos
1.2 eq NaOtBu
Br
3.1 eq tBuONO
5 eq Et3N
toluene/tBuOH (5/1)
+
reflux, 24 h
2) toluene, conc. HCl
THF, relux, o.n.
Boc
H2N
H
N
N
N
N
N
HCl*
HN
HO
N
29 %
quant.
4
5
2.2 eq LiAlH4
THF, reflux
O
24 %
N
N
OH
HO
H2N
N
toluene, 100 °C, o.n.
N
OH
97 %
6
3j
Scheme 4. Synthetic route to homopiperazine compound 3j.
O
O
1.4 eq NaBH(OAc)3
HO
N
HO
1.0 eq AcOH, DCE
H
+
HN
N
N
RT, overnight
26%
HO
HO
8
7
1) SOCl2, cat. DMF, reflux, 3.5 h
2) 1.2 eq
MeO
H2N
N
N
MeO
MeO
O
OH
10% NaOH, DCM, RT, 30 min
53 %
MeO
HN
10
N
N
N
9
1) Boc2O, EtOH, RT, 2.5 h
2) 1.2 eq nBuLi
MeO
MeO
1.1 eq
HN
N
NH
O
MeO
MeO
NH2
THF, 0 °C to RT, then reflux 5 h
25 %
N
11
12
1) CDI, RT, 22 h
2) 1.04 eq
F
HN
N
F
O
O
F
F
OH
N
N
RT, 3 h
20 %
13
14
Scheme 5. Synthesis of test compounds with hydrazone bioisosteres.

2908
N. Mayer et al. / Bioorg. Med. Chem. 23 (2015) 2904–2916
Table 4
ligand binding pocket of ATGL for the first time. These studies indi-
cate that the binding pocket of ATGL is rather sensitive to variations
of the investigated chemotype and that bulky substituents are prob-
lematic, suggesting that the binding pocket is rather narrow and
thin, which might suit an enzyme binding lipids quite well.21,22
Screening data of alternative test compounds produced via Scheme 5
Entry
Compound
IC50 (%)
I200 (%)
1
2
3
4
8
>200
>200
>200
110
20
38
40
57
10
12
14
4. Experimental section
4.1. Materials and methods
phenylpiperazine group. The results in Table 3 immediately sug-
gest that any variation which leads to increased steric bulk by
offering additional substituents or non-sp2-elements (entries 3–7,
9 and 10) were more or less inactive. Only the rather slim 4-fluo-
Acetonitrile, DMF, ethanol and dimethoxyethane were pur-
chased as absolute solvents from Acros Organics, Fisher Scientific
and Sigma Aldrich. Toluene (Sigma Aldrich, 99.7%) was dried in
an aluminum oxide column under inert conditions and stored in
Schlenk bottle over 4A molecule sieves under argon atmosphere.
Tetrahydrofuran was dried at reflux temperature under argon
atmosphere over sodium until benzophenone indicated dryness
by a deep blue color. 1,2-Dichloroethane was purchased from
ACROS Organics as extra dry solvent (99.8%, AcroSealÒ) and
directly used in the reactions. All applied starting materials were
commercially available from Alfa Aesar and Sigma Aldrich and
were used as received. Silica gel chromatography was performed
rophenylpiperazine substrate 3b (IC50 = 50
phenylpiperidino compound 3h (IC50 = 40
l
M, entry 2) and the
lM, entry 8) resulted
in active compounds, although the biological activity was lower
in comparison to parent structure 1h. The data direct to the follow-
ing insights regarding the SAR: the space in the binding pocket
seems to be limited as by introduction of nonplanarity in the case
of cyclohexyl and cyclopentyl ring systems the activity gets lost
and the hydrophobic
to be favorable.
p–p-interaction of an aromatic ring seems
with Acros Organics silica gel 60 (35–70 l
M). 1H and 13C NMR
As the hydrazone moiety is regarded as a problematic molecular
group due to its metabolic instability resulting in toxic side prod-
ucts,12 we prepared compound 8 which can be regarded as a vari-
ant of 1a missing one nitrogen atom (Scheme 5). However, this
compound turned out to be biologically inactive (entry 1,
Table 4). Although hydrazide 10 is a structurally very close analog
to the active hydrazone 1b (Table 1) the hydrazone/hydrazide
switch resulted in an inactive compound (entry 2, Table 4). An
inverso switch of the amido group of 10 led to urea compound
12, which again showed no biological activity against ATGL (entry
3, Table 4). However, when introducing the carbamate moiety
group in 14, which is a structural analog of 1m (entry 13,
spectra were recorded on a Bruker AVANCE III 300 spectrometer
(1H: 300.36 MHz; 13C: 75.53 MHz) and chemical shifts are refer-
enced to residual protonated solvent signals as internal standard.
Electron impact (EI, 70 eV) HRMS spectra were recorded on
Waters GCT Premier equipped with direct insertion (DI) and GC
(HP GC7890A). Melting points were determined with the appara-
tus ‘Mel-TempÒ’ from Electrothermal with an integrated micro-
scopical support.
4.2. General synthesis
Table 1) an active but less potent compound (IC50 = 110 lM, entry
4.2.1. 1-Nitroso-4-phenylpiperazine
4, Table 4) could be generated, which might give rise to a covalent
binding inhibitor based on the intrinsic reactivity of phenol-
carbamates. Taken together these efforts have shown that the
hydrazone moiety is an essential element of the biological activity
of the hit compound 1a and could not be replaced by the most
obvious structural isosteres. In order to further characterize our
hit compound 1a and best inhibitor 1h, these compounds were
tested for ATGL- and HSL-selectivity by the determination of
lipolysis in WAT (white adipose tissue) organ cultures of wild-type,
ATGL-ko, and HSL-ko mice. Although the assay revealed a dose
dependent reduction of FA release, lipolysis was similarly
decreased in ATGL and HSL deficient adipose tissue indicating that
the compounds 1a and 1h show no selectivity between ATGL and
HSL (Suppl. Fig. 1).
A 100 mL Schlenk tube was charged with 1.00 g (940 lL,
6.17 mmol, 1.00 equiv) 1-phenyl piperazine, 50 mL THF and
1.15 mL (9.57 mmol, 1.55 equiv) tert butylnitrite. The mixture
was refluxed overnight. GC–MS analysis indicated full conversion
of the starting material. The orange solution was transferred to a
one-neck round bottom flask and concentrated in vacuum at a
temperature of 30 °C. The crude product was used in the next reac-
tion without further purification. Yield: 813.0 mg (74%), light
brown solid. Rf (MeOH/DCM 1:1) 0.55. 1H NMR (300 MHz,
MeOD): d (ppm) = 7.25–7.20 (m, 2H, Ar-H), 6.96 (d, 3J = 7.8 Hz,
2H, Ar-H), 6.84 (t, 3J = 7.2 Hz, 1H, Ar-H), 3.22 (br s, 4H, 2CH2),
2.82 (br s, 4H, 2CH2). 13C NMR (75.5 MHz, MeOD):
d
(ppm) = 152.3 (Cq), 130.1 (2CHAr), 121.2 (CHAr), 117.6 (2CHAr),
59.2 (2CH2), 50.1 (2CH2). Mp: 36–38 °C.
Further studies revealed that compound 1h does not inhibit
monoglyceride lipase (MGL), or lipoprotein lipase (Suppl. Fig. 2),
but was determined to be toxic (Tox 4 toxicity test, Suppl. Fig. 3)
suggesting that the hydrazone moiety contributes to this outcome.
4.2.2. 4-Phenylpiperazin-1-amine
A 250 mL three-neck round bottom flask with dropping funnel
and reflux condenser was dried under vacuum, filled with nitrogen
and charged with 516 mg (13.6 mmol, 2.20 equiv) LiAlH4 and
25 mL absolute THF. The grey suspension was heated under reflux
and a solution of 1.18 g (6.18 mmol, 1.00 equiv) 1-nitroso-4-
phenylpiperazine in 10 mL absolute THF was added dropwise to
the boiling suspension. After complete addition the suspension
was heated under reflux for further 3 h. GC–MS analysis showed
full conversion of the starting material. The suspension was hydro-
lyzed according to the n,n,3n-method23 (1 mL water, 1 mL 15%
aqueous NaOH and 3 mL water per 1 g LiAlH4) upon which the
color turned to yellow. The mixture was filtrated through a fritted
funnel, the filter cake was washed with 10 mL THF and the filtrate
was concentrated under reduced pressure. Final purification by
3. Conclusion
In summary, we could show that hydrazones resulting from the
condensation of electron rich benzaldehydes with 4-phenylpipera-
zin-1-amine result in quite active inhibitors of ATGL. The best com-
pound 1h exhibits respectable biological activity (IC50 = 10 lM)
considering that the ATGL assay is performed with cell lysate
instead of purified protein. While 1h due to its limited specificity
towards ATGL and intrinsic toxicity will be less useful as molecular
probe than Atglistatin (B), the SAR studies presented in this manu-
script have provided more insight into the characteristics of the

N. Mayer et al. / Bioorg. Med. Chem. 23 (2015) 2904–2916
2909
silica gel filtration (MeOH) yielded the pure product. Yield:
813.0 mg (74%), light brown solid. Rf (MeOH/DCM 1:1) 0.55. 1H
NMR (300 MHz, MeOD): d (ppm) = 7.25–7.20 (m, 2H, Ar-H), 6.96
(d, 3J = 7.8 Hz, 2H, Ar-H), 6.84 (t, 3J = 7.2 Hz, 1H, Ar-H), 3.22 (br s,
4H, 2CH2), 2.82 (br s, 4H, 2CH2). 13C NMR (75.5 MHz, MeOD): d
(ppm) = 152.3 (Cq), 130.1 (2CHAr), 121.2 (CHAr), 117.6 (2CHAr),
59.2 (2CH2), 50.1 (2CH2). Mp: 36–38 °C.
toluene, stirring overnight, recrystallization from 2.5 mL toluene.
Yield: 48.0 mg (68%), brown oil. 1H NMR (300 MHz, DMSO-d6): d
(ppm) = 9.18 (s, 1H, OH), 7.66 (s, 1H, CH@N), 7.26–7.16 (m, 3H,
Ar-H), 7.02–6.97 (m, 3H, Ar-H), 6.83–6.75 (m, 2H, Ar-H), 3.78 (s,
3H, OCH3), 3.31–3.19 (m, 8H, 4CH2). 13C NMR (75.5 MHz, DMSO-
d6): d (ppm) = 150.6 (Cq), 147.7 (Cq-OCH3), 147.0 (Cq-OH), 137.2
(CH@N), 128.9 (2CHAr), 128.1 (Cq), 127.7 (CHAr), 119.9 (CHAr),
115.8 (2CHAr), 115.3 (CHAr), 108.6 (CHAr), 55.4 (OCH3), 51.0
(2CH2), 47.8 (2CH2).
4.2.3. General procedure (GP-1)
An aluminum reaction block was placed on a hotplate stirrer. A
brown 10 mL reaction vessel was charged consecutively with
1.00 equiv 4-phenylpiperazin-1-amine, toluene, 1.00 equiv alde-
hyde and a magnetic stirring bar. The vessel was crimped with a
cap, placed in the preheated (100 °C) reaction block and stirred vig-
orously at 100 °C. GC–MS analysis indicated full conversion of the
starting material. After cooling to rt the cap was removed, the reac-
tion mixture was transferred into a one-neck round bottom flask
and concentrated under reduced pressure to yield the product.
4.2.8. (E)-3-((4-Phenylpiperazin-1-ylimino)methyl)phenol (1e)
According to GP-1: 40.0 mg (226
lmol, 1.00 equiv) 4-phenylpi-
perazin-1-amine, 27.6 mg (226 mol, 1.00 equiv) 3-hydroxyben-
l
zaldehyde, 1.0 mL toluene, stirring overnight. Yield: 32.0 mg
(50%), yellow solid. 1H NMR (300 MHz, DMSO-d6): d (ppm) = 9.41
(s, 1H, OH), 7.64 (s, 1H, CH@N), 7.27–7.21 (m, 2H, Ar-H), 7.15 (t,
3J = 7.8 Hz, 1H, Ar-H), 7.06–6.99 (m, 4H, Ar-H), 6.81 (t, 3J = 7.2 Hz,
1H, Ar-H), 6.68 (dd, 4J = 1.5 Hz, 3J = 7.8 Hz, 1H, Ar-H), 3.32–3.30
(m, 4H, 2CH2), 3.24–3.23 (m, 4H, 2CH2). 13C NMR (75.5 MHz,
DMSO-d6): d (ppm) = 157.4 (Cq-OH), 150.6 (Cq), 137.4 (Cq), 136.1
(CH@N), 129.3 (CHAr), 128.9 (2CHAr), 119.1 (CHAr), 117.3
(CHAr), 115.8 (2CHAr), 115.2 (CHAr), 111.7 (CHAr) 50.6 (2CH2),
47.7 (2CH2). Mp: 190 °C (dec.).
4.2.4. (E)-4-((4-Phenylpiperazin-1-ylimino)methyl)benzene-
1,2-diol (1a)
According to GP-1: 50.0 mg (282
lmol, 1.00 equiv) 4-phenylpi-
perazin-1-amine, 39.0 mg (282 mol, 1.00 equiv) 3,4-dihydroxy-
l
benzaldehyde, 1.0 mL toluene, stirring for 2.5 h. temperature of
30 °C. Yield: 77.0 mg (92%), brown solid. 1H NMR (300 MHz,
DMSO-d6): d (ppm) = 7.58 (s, 1H, CH@N), 7.26–7.21 (m, 2H, Ar-
H), 7.10 (d, 4J = 1.8 Hz, 1H, Ar-H), 7.00 (t, 3J = 8.1 Hz, 2H, Ar-H),
6.83–6.78 (m, 2H, Ar-H), 6.72 (d, 3J = 8.1 Hz, 1H, Ar-H), 3.30–3.28
(m, 4H, 2CH2), 3.17–3.14 (m, 4H, 2CH2). 13C NMR (75.5 MHz,
DMSO-d6): d (ppm) = 150.6 (Cq), 146.0 (Cq-OH), 145.3 (Cq-OH),
137.4 (CH@N), 128.9 (2CHAr), 127.7 (Cq), 119.1 (CHAr), 118.6
(CHAr), 115.8 (2CHAr), 115.3 (CHAr), 112.1 (CHAr), 51.0 (2CH2),
47.8 (2CH2). Mp: 180 °C.
4.2.9. (E)-2-((4-Phenylpiperazin-1-ylimino)methyl)phenol (1f)
According to GP-1: 40.0 mg (226
perazin-1-amine, 27.6 mg (24.0 L, 226
droxybenzaldehyde, 1.0 mL toluene, stirring overnight. Addition
of 14.0 mg (79.0 mol, 0.35 equiv) 4-phenylpiperazin-1-amine
lmol, 1.00 equiv) 4-phenylpi-
l
lmol, 1.00 equiv) 3-hy-
l
after 15 h to reach full conversion of the starting material after fur-
ther 6 h stirring. Yield: 84.0 mg (99%), yellow solid. 1H NMR
(300 MHz, CDCl3): d (ppm) = 11.54 (s, 1H, OH), 7.80 (s, 1H,
CH@N), 7.35–7.16 (m, 4H, Ar-H), 7.06–6.88 (m, 5H, Ar-H), 3.41–
3.38 (m, 8H, 4 CH2). 13C NMR (75.5 MHz, CDCl3): d (ppm) = 157.7
(Cq-OH), 141.6 (CH@N), 129.8 (CHAr), 129.7 (2CHAr), 129.2
(2CHAr), 129.0 (Cq), 120.6 (Cq), 119.1 (CHAr), 118.9 (CHAr),
116.7 (CHAr), 116.6 (CHAr), 51.3 (2CH2), 48.7 (2CH2). Mp: 138–
142 °C.
4.2.5. (E)-N-(3,4-Dimethoxybenzylidene)-4-phenylpiperazin-1-
amine (1b)
According to GP-1: 10.0 mg (56.0
perazin-1-amine, 7.80 mg (47.0 mol, 1.00 equiv) 3,4-dimethoxy-
benzaldehyde, 0.5 mL toluene, stirring for 4 h. Yield: 15.3 mg
(>99%), yellow-brown solid. 1H NMR (300 MHz, CDCl3):
lmol, 1.20 equiv) 4-phenylpi-
l
d
(ppm) = 7.62 (br s, 1H, CH@N), 7.36–7.27 (m, 3H, Ar-H), 7.05–6.98
(m, 3H, Ar-H), 6.93–6.84 (m, 2H, Ar-H), 3.94 (s, 3H, OCH3), 3.90 (s,
3H, OCH3), 3.40–3.38 (m, 4H, 2CH2), 3.34–3.32 (m, 4H, 2CH2). 13C
NMR (75.5 MHz, CDCl3): d (ppm) = 150.9 (2Cq-OCH3), 149.6 (Cq),
149.3 (Cq), 137.3 (CH@N), 129.2 (2CHAr), 120.4 (CHAr), 120.2
(CHAr), 116.5 (2CHAr), 110.7 (CHAr), 107.5 (CHAr), 55.9 (OCH3),
55.8 (OCH3), 51.4 (2CH2), 48.9 (2CH2). Mp: 153–154 °C.
4.2.10. (E)-4-((4-Phenylpiperazin-1-ylimino)methyl)phenol (1g)
According to GP-1: 40.0 mg (226
lmol, 1.00 equiv) 4-phenylpi-
perazin-1-amine, 27.6 mg (226 mol, 1.00 equiv) 4-hydroxyben-
l
zaldehyde, 1.0 mL toluene, stirring overnight. Yield: 63.5 mg
(>99%), beige solid. 1H NMR (300 MHz, DMSO-d6): d (ppm) = 9.59
(s, 1H, OH), 7.66 (s, 1H, CH@N), 7.42 (d, 3J = 8.7 Hz, 2H, Ar-H),
7.25–7.20 (m, 2H, Ar-H), 7.00 (d, 3J = 8.1 Hz, 2H, Ar-H), 6.83–6.74
(m, 3H, Ar-H), 3.30–3.28 (m, 4H, 2CH2), 3.18–3.16 (m, 4H, 2CH2).
13C NMR (75.5 MHz, DMSO-d6): d (ppm) = 157.6 (Cq-OH), 150.6
(Cq), 137.1 (CH@N), 128.9 (2CHAr), 127.3 (2CHAr), 127.2 (Cq),
119.1 (CHAr), 115.7 (2CHAr), 115.3 (2CHAr), 51.0 (2CH2), 47.8
(2CH2). Mp: 192 °C (dec.).
4.2.6. (E)-2-Methoxy-5-((4-phenylpiperazin-1-ylimino)methyl)-
phenol (1c)
According to GP-1: 10.0 mg (56.0
perazin-1-amine, 7.80 mg (51 mol, 1.00 equiv) 3-hydrox-
yanisaldehyde, 0.5 mL toluene, stirring for 4.5 h. Yield: 16.0 mg
(>99%), yellow solid. 1H NMR (300 MHz, DMSO-d6):
lmol, 1.10 equiv) 4-phenylpi-
l
d
(ppm) = 9.06 (s, 1H, OH), 7.62 (s, 1H, CH@N), 7.26–7.21 (m, 2H,
Ar-H), 7.12 (d, 4J = 1.5 Hz, 1H, Ar-H), 7.02–6.79 (m, 5H, Ar-H),
3.77 (s, 3H, OCH3), 3.31–3.29 (m, 4H, 2CH2), 3.20–3.18 (m, 4H,
2CH2). 13C NMR (75.5 MHz, DMSO-d6): d (ppm) = 150.6 (Cq),
148.0 (Cq-OCH3), 146.5 (Cq-OH), 136.7 (CH@N), 129.2 (Cq), 128.9
(2CHAr), 119.1 (CHAr), 118.3 (CHAr), 115.8 (2CHAr), 111.8
(CHAr), 111.7 (CHAr) 55.5 (OCH3), 50.9 (2CH2), 47.8 (2CH2). Mp:
180 °C (dec.).
4.2.11. (E)-4-(4-Phenylpiperazin-1-ylimino)methylbenzene-1,3-
diol (1h)
According to GP-1: 30.0 mg (169
lmol, 1.00 equiv) 4-phenylpi-
perazin-1-amine, 23.4 mg (169 mol, 1.00 equiv) 2,4-dihydroxy-
l
benzaldehyde), 1.0 mL toluene, stirring for 2 h. Yield: 51.0 mg
(>99%), brown solid. Rf (MeOH): 0.62. 1H NMR (300 MHz, DMSO-
d6): d (ppm) = 11.60 (s, 1H, OH), 9.72 (br s, 1H, OH), 7.95 (s, 1H,
CH@N), 7.26–7.16 (m, 3H, Ar-H), 7.00 (d, 3J = 8.1 Hz, 2H, Ar-H),
6.81 (t, 3J = 7.2 Hz, 1H, Ar-H), 6.32 (dd, 3J = 8.1 Hz, 4J = 2.1 Hz, 1H,
Ar-H), 6.25 (d, 4J = 2.1 Hz, 1H, Ar-H), 3.33–3.30 (m, 4H, 2CH2),
4.2.7. (E)-2-Methoxy-4-((4-phenylpiperazin-1-ylimino)methyl)-
phenol (1d)
3.18–3.15 (m, 4H, 2CH2). 13C NMR (75.5 MHz, DMSO-d6):
d
According to GP-1: 44.0 mg (249
lmol, 1.00 equiv) 4-phenylpi-
(ppm) = 159.0 (Cq-OH), 158.6 (Cq-OH), 150.5 (Cq), 142.3 (CH@N),
perazin-1-amine, 31.3 mg (205 mol, 1.00 equiv) vanilline, 1.0 mL
l
130.7 (CHAr), 128.9 (2CHAr), 119.2 (CHAr), 115.8 (2CHAr), 111.4

2910
N. Mayer et al. / Bioorg. Med. Chem. 23 (2015) 2904–2916
(Cq), 107.0 (CHAr), 102.4 (CHAr), 51.1 (2CH2), 47.5 (2CH2). Mp:
(Cq), 129.2 (2CHAr), 120.3 (CHAr), 116.6 (2CHAr), 103.2 (CHAr),
183–184 °C.
60.9 (OCH3), 56.1 (2OCH3), 51.2 (2CH2), 48.9 (2CH2). Mp: 93–98 °C.
4.2.12. (E)-4-Phenyl-N-(4-propoxybenzylidene)piperazin-1-
amine (1i)
4.2.16. (E)-N-(3,4-Difluorobenzylidene)-4-phenylpiperazin-1-
amine (1m)
According to GP-1: 30.0 mg (169
perazin-1-amine, 27.7 mg (27.0
l
mol, 1.00 equiv) 4-phenylpi-
According to GP-1: 250 mg (1.41 mmol, 2.00 equiv) 4-phenylpi-
l
L, 169 mol, 1.00 equiv) 4-
l
perazin-1-amine, 100 mg (78.0
fluorobenzaldehyde, 3.0 mL toluene, stirring overnight. Yield:
189.8 g (90%), yellow solid. 1H NMR (300 MHz, CDCl3):
lL, 706 lmol, 1.00 equiv) 3,4-di-
propoxybenzaldehyde, 1.0 mL toluene, stirring overnight. Yield:
40.0 mg (71%), light yellow solid. 1H NMR (300 MHz, CDCl3): d
(ppm) = 7.65 (s, 1H, CH@N), 7.57 (d, 3J = 8.7 Hz, 2H, Ar-H), 7.33–
7.28 (m, 2H, Ar-H), 6.99 (d, 3J = 8.1 Hz, 2H, Ar-H), 6.93–6.88 (m,
3H, Ar-H), 3.94 (t, 3J = 6.6 Hz, 2H, OCH2), 3.41–3.38 (m, 4H,
2CH2), 3.33–3.30 (m, 4H, 2CH2), 1.82 (sext, 3J = 6.9 Hz, 3J = 7.2 Hz,
2H, CH2), 1.05 (t, 3J = 7.2 Hz, 3H, CH3). 13C NMR (75.5 MHz,
CDCl3): d (ppm) = 159.6 (Cq-OCH2), 150.9 (Cq), 137.7 (CH@N),
129.2 (2CHAr), 128.6 (Cq), 127.6 (2CHAr), 120.2 (CHAr), 116.5
(2CHAr), 114.6 (2CHAr), 69.5 (OCH2), 51.5 (2CH2), 48.9 (2CH2),
22.6 (CH2), 10.5 (CH3). Mp: 184–185 °C.
d
(ppm) = 7.52–7.43 (m, 2H, CH@N, Ar-H), 7.31–7.24 (m, 3H, Ar-H),
7.16–7.07 (m, 1H, Ar-H), 6.98 (d, 3J = 7.8 Hz, 2H, Ar-H), 6.90 (t,
3J = 7.2 Hz, 1H, Ar-H), 3.37–3.36–3.34 (m, 8H, 4CH2). 13C NMR
(75.5 MHz, CDCl3): d (ppm) = 152.4, 152.2 (3JC–F = 16.5 Hz, Cq),
151.8, 149.1 (1JC–F = 203.2 Hz, Cq-F), 150.8, 148.7 (1JC–F = 161.2 Hz,
Cq-F), 133.6 (CH@N), 133.5 (Cq), 129.2 (2CHAr), 122.5, 122.4,
4
122.4, 122.3 (3JC–F = 6.2 Hz, JC–F = 3.2 Hz, CHAr), 120.4 (CHAr),
117.3, 117.1 (2JC–F = 17.6 Hz, CHAr), 116.6 (2CHAr), 114.2, 114.0
(2JC–F = 18.2 Hz, CHAr), 51.0 (2CH2), 48.9 (2CH2). Mp: 152–154 °C.
HRMS (EI+): m/z: calcd for C17H17N3F2 [M]+: 301.1391; found
301.1395.
4.2.13. (E)-N-(4-Phenoxybenzylidene)-4-phenylpiperazin-1-
amine (1j)
According to GP-1: 40.0 mg (226
perazin-1-amine, 44.7 mg (226 mol, 1.00 equiv) 4-phenoxyben-
zaldehyde, 1.0 mL toluene, stirring overnight. Yield: 76.0 mg
(94%), yellow-brown solid. 1H NMR (300 MHz, CDCl3):
l
mol, 1.00 equiv) 4-phenylpi-
4.2.17. (E)-N-(3,4-Dichlorobenzylidene)-4-phenylpiperazin-1-
amine (1n)
l
According to GP-1: 50.0 mg (282
perazin-1-amine, 25.0 mg (141 mol, 1.00 equiv) 3,4-dichloroben-
zaldehyde, 1.0 mL toluene, stirring overnight. Addition of another
14.0 mg (80.0 mol, 0.60 equiv) 4-phenylpiperazin-1-amine after
lmol, 2.00 equiv) 4-phenylpi-
d
l
(ppm) = 7.66 (s, 1H, CH@N), 7.61 (d, 3J = 8.7 Hz, 2H, Ar-H), 7.38–
7.28 (m, 4H, Ar-H), 7.12 (t, 3J = 7.2 Hz, 1H, Ar-H), 7.02 (m, 6H, Ar-
H), 6.92 (t, 3J = 7.2 Hz, 1H, Ar-H), 3.41–3.34 (m, 8H, 4 CH2). 13C
NMR (75.5 MHz, CDCl3): d (ppm) = 157.6 (Cq), 156.9 (Cq), 150.9
(Cq), 136.4 (CH@N), 131.2 (Cq), 129.8 (2CHAr), 129.2 (2CHAr),
127.7 (2CHAr), 123.4 (2CHAr), 120.3 (CHAr), 119.0 (2CHAr), 118.8
(2CHAr), 116.6 (CHAr), 51.3 (2CH2), 48.9 (2CH2). Mp: 161–164 °C.
HRMS (EI+): m/z: calcd for C23H23ON3 [M]+: 357.1841; found
357.1844.
l
16 h to reach full conversion of the starting material after further
stirring over a second night. Yield: 67.0 mg (90%), light yellow
solid. 1H NMR (300 MHz, CDCl3): d (ppm) = 7.73 (s, 1H, CH@N),
7.47 (s, 1H, Ar-H), 7.42 (s, 2H, Ar-H), 7.34–7.28 (m, 2H, Ar-H),
7.00 (d, 3J = 8.1 Hz, 2H, Ar-H), 6.95–6.90 (m, 1H, Ar-H), 3.38 (s,
8H, 4CH2). 13C NMR (75.5 MHz, CDCl3): d (ppm) = 150.7 (Cq),
136.3 (CH@N), 133.0 (Cq-Cl), 132.8 (Cq), 131.6 (Cq-Cl), 130.4
(CHAr), 129.2 (2CHAr), 127.5 (CHAr), 125.2 (CHAr), 120.5 (CHAr),
116.7 (2CHAr), 50.8 (2CH2), 49.0 (2CH2). Mp: 176–178 °C.
4.2.14. (E)-4-Phenyl-N-(2,4,5-trimethoxybenzyliden)piperazin-
1-amine (1k)
According to GP-1: 30.0 mg (169
perazin-1-amine, 33.1 mg (169
trimethoxybenzaldehyde, 1.0 mL toluene, stirring overnight.
Addition of 3.0 mg (15.0 mol, 0.09 equiv) 4-phenylpiperazin-1-
l
l
mol, 2.00 equiv) 4-phenylpi-
mol, 1.00 equiv) 2,4,5-
4.2.18. (E)-N-(2,4-Dichlorobenzylidene)-4-phenylpiperazin-1-
amine (1o)
According to GP-1: 50.0 mg (282
perazin-1-amine, 25.0 mg (141 mol, 1.00 equiv) 2,4-dichloroben-
zaldehyde, 1.0 mL toluene, stirring overnight. Addition of another
14.0 mg (80.0 mol, 0.60 equiv) 4-phenylpiperazin-1-amine after
lmol, 2.00 equiv) 4-phenylpi-
l
l
amine after 18 h to reach full conversion of the starting material
after further stirring over a second night. Yield: 55.0 mg (83%), yel-
low-orange solid. 1H NMR (300 MHz, CDCl3): d (ppm) = 8.00 (br s,
1H, CH@N), 7.46 (s, 1H, Ar-H), 7.34–7.27 (m, 3H, Ar-H), 6.99 (d,
3J = 7.8 Hz, 2H, Ar-H), 6.50 (s, 1H, arom.H), 3.91 (s, 3H, OCH3),
3.90 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.41–3.38 (m, 4H, 2CH2),
l
16 h to reach full conversion of the starting material after further
stirring over a second night. Yield: 71.0 mg (95%), yellow solid.
1H NMR (300 MHz, CDCl3): d (ppm) = 7.85 (d, 3J = 8.7 Hz, 1H, Ar-
H), 7.77 (s, 1H, CH@N), 7.29 (d, 4J = 2.1 Hz, 1H, Ar-H), 7.25–7.13
(m, 3H, Ar-H), 6.94–6.91 (m, 2H, Ar-H), 6.85 (t, 3J = 7.2 Hz, 1H,
3.34–3.31 (m, 4H, 2CH2). 13C NMR (75.5 MHz, CDCl3):
d
(ppm) = 152.1 (Cq-OCH3), 151.0 (Cq-OCH3), 150.3 (Cq-OCH3),
143.7 (CH@N), 129.1 (2CHAr), 128.2 (Cq), 120.1 (CHAr), 116.6
(Cq), 116.5 (2CHAr), 107.9 (CHAr), 97.3 (CHAr), 56.7 (OCH3), 56.2
(OCH3), 56.0 (OCH3), 51.5 (2CH2), 48.9 (2CH2). Mp: 153–156 °C.
Ar-H), 3.33 (s, 8H, 4 d
CH2). 13C NMR (75.5 MHz, CDCl3):
(ppm) = 150.8 (Cq), 133.9 (Cq-Cl), 133.1 (Cq), 132.1 (Cq-Cl), 130.9
(CH@N), 129.3 (CHAr), 129.2 (2CHAr), 127.3 (CHAr), 127.1
(CHAr), 120.4 (CHAr), 116.7 (2CHAr), 50.9 (2CH2), 48.9 (2CH2).
Mp: 113–115 °C.
4.2.15. (E)-4-Phenyl-N-(3,4,5-trimethoxybenzylidene)piperazin-
1-amine (1l)
4.2.19. (E)-N-(2-Nitrobenzylidene)-4-phenylpiperazin-1-amine
According to GP-1: 30.0 mg (169
perazin-1-amine, 33.1 mg (169
trimethoxybenzaldehyde, 1.0 mL toluene, stirring overnight.
Addition of 2.00 mg (10.0 mol, 0.06 equiv) 4-phenylpiperazin-1-
amine after 18 h to reach full conversion of the starting material
after further stirring over a second night. Yield: 28.0 mg (44%), yel-
low solid. 1H NMR (300 MHz, CDCl3): d (ppm) = 7.93 (s, 1H, CH@N),
7.70–7.64 (m, 3H, Ar-H), 7.37 (d, 3J = 8.1 Hz, 2H, Ar-H), 7.33–7.28
(m, 2H, Ar-H), 4.28 (s, 6H, 2OCH3), 4.24 (s, 3H, OCH3), 3.78–3.71
(m, 8H, 4 CH2). 13C NMR (75.5 MHz, CDCl3): d (ppm) = 153.4
(2Cq-OCH3), 150.9 (Cq-OCH3), 138.5 (Cq), 136.4 (CH@N), 131.7
l
l
mol, 2.00 equiv) 4-phenylpi-
mol, 1.00 equiv) 3,4,5-
(1p)
According to GP-1: 40.0 mg (226
perazin-1-amine, 26.2 mg (226 mol, 1.00 equiv) 2-nitroben-
zaldehyde, 1.0 mL toluene, stirring overnight. Addition of 3.40 mg
(22.0 mol, 0.10 equiv) 2-nitrobenzaldehyde after 20 h to reach
full conversion of the starting material after further 3 h stirring.
Yield: 60.0 mg (97%), yellow-brown solid. 1H NMR (300 MHz,
CDCl3): d (ppm) = 8.15–8.13 (m, 1H, Ar-H), 8.10 (s, 1H, CH@N),
8.00 (d, 3J = 8.1 Hz, 1H, Ar-H), 7.58 (t, 3J = 7.8 Hz, 1H, Ar-H), 7.41–
7.28 (m, 3H, Ar-H), 7.01 (d, 3J = 7.8 Hz, 2H, Ar-H), 6.92 (t,
3J = 7.2 Hz, 1H, Ar-H), 3.45–3.39 (m, 8H, 4CH2). 13C NMR
lmol, 1.00 equiv) 4-phenylpi-
l
l
l

N. Mayer et al. / Bioorg. Med. Chem. 23 (2015) 2904–2916
2911
(75.5 MHz, CDCl3): d (ppm) = 150.8 (Cq), 147.3 (Cq-NO2), 133.0
4.2.24. (E)-N-(4-(Dimethylamino)benzylidene)-4-phenylpi-
(CH@N), 131.1 (Cq), 130.2 (CHAr), 129.2 (2CHAr), 127.9 (CHAr),
127.5 (CHAr), 124.6 (CHAr), 120.4 (CHAr), 116.6 (2CHAr), 50.8
(2CH2), 48.8 (2CH2). Mp: 103–106 °C.
perazin-1-amine (1u)
According to GP-1: 40.0 mg (226
l
mol, 1.00 equiv) 4-phenylpi-
perazin-1-amine, 34.0 mg (226
laminobenzaldehyde, 1.0 mL toluene, stirring overnight. Addition
of another 4.00 mg (23.0 mol, 0.10 equiv) 4-phenylpiperazin-1-
lmol, 1.00 equiv) 4-dimethy-
4.2.20. (E)-N-(2-Bromobenzylidene)-4-phenylpiperazin-1-amine
l
(1q)
amine after 18 h to reach full conversion of the starting material
after further 2 h stirring. Yield: 43.0 mg (61%), light yellow solid.
1H NMR (300 MHz, CDCl3): d (ppm) = 7.68 (br s, 1H, CH@N), 7.53
(d, 3J = 8.7 Hz, 2H, Ar-H), 7.33–7.28 (m, 2H, Ar-H), 7.00 (d,
3J = 8.1 Hz, 2H, Ar-H), 6.90 (t, 3J = 7.2 Hz, 1H, Ar-H), 6.71 (d,
3J = 8.7 Hz, 2H, Ar-H), 3.40–3.38 (m, 4H, 2CH2), 3.31–3.29 (m, 4H,
According to GP-1: 40.0 mg (226
perazin-1-amine, 34.8 mg (22.0 L, 118
l
mol, 1.20 equiv) 4-phenylpi-
l
lmol, 1.00 equiv) 2-bro-
mobenzaldehyde, 1.0 mL toluene, stirring overnight. Yield:
69.0 mg (89%), yellow-orange solid. 1H NMR (300 MHz, CDCl3): d
(ppm) = 7.95 (dd, 3J = 7.8 Hz, 4J = 1.5 Hz, 1H, Ar-H), 7.90 (s, 1H,
CH@N), 7.53 (dd, 3J = 8.1 Hz, 4J = 0.9 Hz, 1H, Ar-H), 7.33–7.28 (m,
3H, Ar-H), 7.16–7.10 (m, 1H, Ar-H), 7.00 (d, 3J = 7.8 Hz, 2H, Ar-H),
6.92 (t, 3J = 7.5 Hz, 1H, Ar-H), 3.41 (s, 8H, 4 CH2). 13C NMR
(75.5 MHz, CDCl3): d (ppm) = 150.9 (Cq), 135.0 (CH@N), 134.8
(Cq), 132.8 (CHAr), 129.3 (CHAr), 129.2 (2CHAr), 127.5 (CHAr),
126.7 (CHAr), 123.2(Cq-Br), 120.3 (CHAr), 116.6 (2CHAr), 51.0
(2CH2), 48.9 (2CH2). Mp: 88–90 °C.
2CH2), 2.99 (s, 6H, 2CH3). 13C NMR (75.5 MHz, CDCl3):
d
(ppm) = 151.0 (Cq-N), 150.8 (Cq), 139.5 (CH@N), 129.1 (2CHAr),
127.6 (2CHAr), 124.2 (Cq), 120.0 (CHAr), 116.4 (2CHAr), 112.1
(2CHAr), 51.8 (2CH2), 48.9 (2CH2), 40.4 (2CH3). Mp: 190 °C (dec.).
4.2.25. (E)-N-(4-((4-Phenylpiperazin-1-ylimino)methyl)phenyl)-
acetamide (1v)
According to GP-1: 30.0 mg (169
lmol, 1.00 equiv) 4-phenylpi-
4.2.21. (E)-3-((4-Phenylpiperazin-1-ylimino)methyl)benzonitrile
perazin-1-amine, 27.5 mg (169 mol, 1.00 equiv) 4-acetamidoben-
l
(1r)
zaldehyde, 1.0 mL toluene, stirring overnight. Yield: 54.0 mg (96%),
yellow solid. 1H NMR (300 MHz, DMSO-d6): d (ppm) = 10.00 (s, 1H,
NH), 7.68 (s, 1H, CH@N), 7.61–7.50 (m, 4H, Ar-H), 7.26–7.21 (m,
2H, Ar-H), 7.00 (d, 3J = 8.1 Hz, 2H, Ar-H), 6.81 (t, 3J = 7.2 Hz, 1H,
Ar-H), 3.32–3.30 (m, 4H, 2CH2), 3.23–3.21 (m, 4H, 2CH2), 2.05 (s,
3H, CH3). 13C NMR (75.5 MHz, DMSO-d6): d (ppm) = 168.1 (C@O),
150.6 (Cq), 139.1 (Cq-NH), 136.0 (CH@N), 130.9 (Cq), 128.8
(2CHAr), 126.2 (2CHAr), 119.1 (CHAr), 118.8 (2CHAr), 115.8
(2CHAr), 50.7 (2CH2), 47.7 (2CH2), 23.9 (CH3). Mp: 229–232 °C.
HRMS (EI+): m/z: calcd for C19H22ON4 [M]+: 322.1794; found
322.1795.
According to GP-1: 40.0 mg (226
perazin-1-amine, 30.0 mg (226 mol, 1.00 equiv) 3-formylben-
lmol, 1.00 equiv) 4-phenylpi-
l
zonitrile, 1.0 mL toluene, stirring for 6 h. Yield: 67.0 mg (99%),
yellow solid. 1H NMR (300 MHz, CDCl3): d (ppm) = 7.91 (s, 1H,
CH@N), 7.82 (dd, 3J = 7.8 Hz, 4J = 1.2 Hz, 1H, Ar-H), 7.55–7.52 (m,
2H, Ar-H), 7.44 (t, 3J = 7.8 Hz, 1H, Ar-H), 7.33–7.28 (m, 2H, Ar-H),
7.99 (d, 3J = 7.8 Hz, 2H, Ar-H), 6.92 (t, 3J = 7.2 Hz, 1H, Ar-H), 3.39
(s, 8H, 4 CH2). 13C NMR (75.5 MHz, CDCl3): d (ppm) = 150.8 (Cq),
137.5 (CH@N), 132.6 (CHAr), 131.0 (Cq), 130.0 (CHAr), 129.4
(CHAr), 129.3 (CHAr), 129.2 (2CHAr), 120.4 (CHAr), 118.8 (CN),
116.7 (2CHAr), 112.7 (Cq-CN), 50.8 (2CH2), 48.9 (2CH2). Mp:
140–142 °C. HRMS (EI+): m/z: calcd for C18H18N4 [M]+: 290.1531;
found 290.1539.
4.2.26. (E)-2-(Methyl(4-((4-phenylpiperazin-1-ylimino)methyl)-
phenyl)amino)ethanol (1w)
According to GP-1: 30.0 mg (169
lmol, 1.00 equiv) 4-phenylpi-
4.2.22. (E)-4-((4-Phenylpiperazin-1-ylimino)methyl)benzonitrile
(1s)
perazin-1-amine, 30.3 mg (169 mol, 1.00 equiv) N-methyl-N-(2-
l
hydroxyethyl)-4-aminobenzaldehyde, 1.0 mL toluene, stirring
overnight. Yield: 48.0 mg (84%), light yellow solid. 1H NMR
(300 MHz, DMSO-d6): d (ppm) = 7.66 (s, 1H, CH@N), 7.42 (d,
3J = 8.7 Hz, 2H, Ar-H), 7.23 (t, 3J = 8.1 Hz, 2H, Ar-H), 6.99 (d,
3J = 8.1 Hz, 2H, Ar-H), 6.81 (t, 3J = 7.2 Hz, 1H, Ar-H), 6.68 (d,
3J = 9.0 Hz, 2H, Ar-H), 4.68 (t, 3J = 5.1 Hz, OH), 3.58–3.52 (m, 2H,
CH2), 3.43–3.39 (m, 2H, CH2), 3.32–3.38 (m, 4H, 2CH2), 3.17–3.14
(m, 4H, 2CH2), 2.95 (s, 3H, N-CH3). 13C NMR (75.5 MHz, DMSO-
d6): d (ppm) = 150.6 (Cq-N), 149.2 (Cq), 138.0 (CH@N), 128.8
(2CHAr), 127.1 (2CHAr), 123.4 (Cq), 119.0 (CHAr), 115.7 (2CHAr),
111.3 (2CHAr), 58.0 (N-CH2), 54.0 (O-CH2), 51.2 (2CH2), 47.8
(2CH2), 38.5 (NCH3). Mp: 202–204 °C. HRMS (EI+): m/z: calcd for
According to GP-1: 40.0 mg (226
lmol, 1.00 equiv) 4-phenylpi-
perazin-1-amine, 29.6 mg (226 mol, 1.00 equiv) 4-formylben-
l
zonitrile, 1.0 mL toluene, stirring overnight. Yield: 60.0 mg (92%),
yellow-orange solid. 1H NMR (300 MHz, CDCl3): d (ppm) = 7.69
(d, 3J = 8.4 Hz, 2H, Ar-H), 7.61 (d, 3J = 8.4 Hz, 2H, Ar-H), 7.52 (s,
1H, CH@N), 7.33–7.28 (m, 2H, Ar-H), 6.99 (d, 3J = 7.8 Hz, 2H, Ar-
H), 6.92 (t, 3J = 7.2 Hz, 1H, Ar-H), 3.40 (s, 8H, 4 CH2). 13C NMR
(75.5 MHz, CDCl3):
d (ppm) = 150.8 (Cq), 140.6 (Cq), 132.5
(CH@N), 132.3 (2CHAr), 129.2 (2CHAr), 126.2 (2CHAr), 120.5
(CHAr), 119.1 (CN),116.7 (2CHAr), 110.8 (Cq-CN), 50.7 (2CH2),
48.9 (2CH2). Mp: 165–169 °C. HRMS (EI+): m/z: calcd for
C18H18N4 [M]+: 290.1531; found 290.1519.
C
20H26ON4 [M]+: 338.2107; found 338.2117.
4.2.23. (E)-N-(4-Methylbenzylidene)-4-phenylpiperazin-1-amine
(1t)
4.2.27. (E)-N-(Benzo[d][1,3]dioxol-5-ylmethylen)-4-phenylpi-
perazin-1-amine (1x)
According to GP-1: 40.0 mg (226
perazin-1-amine, 27.0 mg (27.0 L, 226
benzaldehyde, 1.0 mL toluene, stirring overnight. Yield: 64.0 mg
lmol, 1.00 equiv) 4-phenylpi-
According to GP-1: 38.0 mg (215
lmol, 1.00 equiv) 4-phenylpi-
l
l
mol, 1.00 equiv) p-tolyl-
perazin-1-amine, 1.0 mL toluene, 32.2 mg (215
l
mol, 1.00 equiv)
benzo[d][1,3]-dioxol-5-carbaldehyde. Stirring at 100 °C for 4 h.
Final purification by recrystallization from 1.0 mL toluene pro-
duced the pure product. Yield: 40.0 mg (67%), orange-brown solid.
1H NMR (300 MHz, DMSO-d6): d (ppm) = 7.67 (s, 1H, CH@N), 7.26–
7.21 (m, 2H, Ar-H), 7.18–7.17 (m, 1H, Ar-H), 7.05–6.99 (m, 3H, Ar-
H), 6.92–6.90 (m, 1H, Ar-H), 6.83–6.79 (m, 1H, Ar-H), 6.03 (s, 2H,
CH2), 3.31 (m, 4H, 2CH2), 3.21 (m, 4H, 2CH2). 13C NMR
(75.5 MHz, DMSO-d6): d (ppm) = 150.6 (Cq), 147.6 (Cq), 147.2
(Cq), 136.1 (CH@N), 130.7 (Cq), 128.8 (2CHAr), 121.0 (CHAr),
(99%), light yellow solid. 1H NMR (300 MHz, CDCl3):
d
(ppm) = 7.66 (s, 1H, CH@N), 7.53 (d, 3J = 8.1 Hz, 2H, Ar-H), 7.30
(dd, 3J = 8.4 Hz, 3J = 7.5 Hz, 2H, Ar-H), 7.17 (d, 3J = 7.8 Hz, 2H, Ar-
H), 7.00 (d, 3J = 8.1 Hz, 2H, Ar-H), 6.91 (t, 3J = 7.5 Hz, 1H, Ar-H),
3.41–3.34 (m, 8H,
4 d
CH2). 13C NMR (75.5 MHz, CDCl3):
(ppm) = 150.9 (Cq), 138.4 (C-CH3), 137.3 (CH@N), 133.2 (Cq),
129.3 (2CHAr), 129.2 (2CHAr), 126.2 (2CHAr), 120.3 (CHAr),
116.6 (2CHAr), 51.3 (2CH2), 48.9 (2CH2), 21.3 (CH3). Mp: 184–
186 °C.

2912
N. Mayer et al. / Bioorg. Med. Chem. 23 (2015) 2904–2916
119.1 (CHAr), 115.8 (2CHAr), 108.1 (CHAr), 104.3 (CHAr), 101.0
(CH2), 50.8 (2CH2), 47.7 (2CH2). Mp: 125–129 °C.
(2CH2), 34.7 (CH2), 22.1 (CH2), 13.6 (CH3). Mp: 48 °C. HRMS (EI+):
m/z: calcd for C16H23N3 [M]+: 257.1892; found 257.1898.
4.2.28. (E)-N-((2,3-Dihydrobenzol[b][1,4]dioxin-6-yl)methylen)-
4-phenylpiperazin-1-amine (1y)
4.2.32. (E)-N-(Cyclohexylmethylen)-4-phenylpiperazin-1-amine
(2c)
According to GP-1: 50.0 mg (282
lmol, 1.00 equiv) 4-phenylpi-
According to GP-1: 40.0 mg (226
l
mol, 1.00 equiv) 4-phenylpi-
mol, 1.00 equiv) cyclo-
perazin-1-amine, 46.0 mg (282 mol, 1.00 equiv) 1,4-benzodiox-
l
perazin-1-amine, 25.1 mg (27.1 L, 226
l
l
an-6-carboxaldehyde, 1.0 mL toluene, stirring overnight. Yield:
87.0 mg (95%), light brown solid. 1H NMR (300 MHz, DMSO-d6): d
(ppm) = 7.63 (s, 1H, CH@N), 7.26–7.21 (m, 2H, Ar-H), 7.09–7.06
(m, 2H, Ar-H), 7.01–6.98 (m, 2H, Ar-H), 6.86–6.79 (m, 2H, Ar-H),
4.24 (s, 4H, 2CH2), 3.32–3.29 (m, 4H, 2CH2), 3.21–3.17 (s, 4H,
2CH2). 13C NMR (75.5 MHz, DMSO-d6): d (ppm) = 150.6 (Cq-O),
143.5 (Cq), 143.3 (Cq-O), 135.9 (CH@N), 129.7 (Cq), 128.8
(2CHAr), 119.1 (CHAr), 117.0 (CHAr), 115.8 (2CHAr), 114.0
(CHAr), 64.0 (2CH2), 50.8 (2CH2), 47.7 (2CH2). Mp: 186–187 °C.
HRMS (EI+): m/z: calcd for C19H21O2N3 [M]+: 323.1634; found
323.1644.
hexanecarboxaldehyde, 1.0 mL toluene, stirring for 4 h. Yield:
60.0 mg (98%), yellow-brown solid. 1H NMR (300 MHz, CDCl3): d
(ppm) = 7.31–7.24 (m, 3H, Ar-H, CH@N), 6.96 (d, 3J = 8.1 Hz, 2H,
Ar-H), 6.90–6.86 (m, 1H, Ar-H), 3.35–3.32 (m, 4H, 2CH2), 3.12–
3.08 (m, 4H, 2CH2), 2.25–2.21 (m, 1H, CH), 1.82–1.66 (m, 5H,
CH2, CH), 1.35–1.16 (m, 5H, CH2, CH). 13C NMR (75.5 MHz,
CDCl3): d (ppm) = 150.9 (CH@N), 147.4 (Cq), 129.1 (2CHAr), 120.0
(CHAr), 116.4 (2CHAr), 51.8 (2CH2), 48.8 (2CH2), 41.4 (CH), 31.0
(2CH2), 26.0 (CH2), 25.6 (2CH2). Mp: 69–71 °C. HRMS (EI+): m/z:
calcd for C17H25N3 [M]+: 271.2048; found 271.2058.
4.2.33. (E)-N-(Cyclohexenylmethylen)-4-phenylpiperazin-1-amine
(2d)
4.2.29. (E)-5-((4-Phenylpiperazin-1-ylimino)methyl)-1H-benzo-
[d]imidazol-2(3H)-one (1z)
According to GP-1: 40.0 mg (226
l
mol, 1.00 equiv) 4-phenylpi-
mol, 1.00 equiv) 1-cyclo-
According to GP-1: 65.6 mg (370
perazin-1-amine, 1.0 mL toluene, 60.0 mg (370
2-oxo-2,3-dihydro-1H-benzimidazol-5-carbaldehyde. Stirring at
100 °C for 5 h. Additional 6.10 mg (34.0 mol, 0.09 equiv) 4-phe-
l
mol, 1.00 equiv) 4-phenylpi-
perazin-1-amine, 24.9 mg (25.8 L, 226
l
l
lmol, 1.00 equiv)
hexene-1-carboxaldehyde, 1.0 mL toluene, stirring for 4 h. Yield:
57.0 mg (94%), yellow-brown solid. 1H NMR (300 MHz, CDCl3): d
(ppm) = 7.34 (s, 1H, CH@N), 7.31–7.26 (m, 2H, Ar-H), 6.97 (d,
3J = 8.1 Hz, 2H, Ar-H), 6.88 (t, 3J = 7.2 Hz, 1H, Ar-H), 5.92 (t,
3J = 3.9 Hz, 1H, CH), 3.36–3.33 (m, 4H, 2CH2), 3.20–3.17 (m, 4H,
2CH2), 2.31 (br s, 2H, CH2), 2.19 (br s, 2H, CH2), 1.67–1.65 (m,
4H, 2CH2). 13C NMR (75.5 MHz, CDCl3): d (ppm) = 151.9 (Cq),
142.5 (CH@N), 136.3 (Cq), 132.3 (CH), 129.1 (2CHAr), 120.0
(CHAr), 116.4 (2CHAr), 51.5 (2CH2), 48.9 (2CH2), 25.9 (CH2), 23.8
(CH2), 22.7 (CH2), 22.2 (CH2). Mp: 118–120 °C. HRMS (EI+): m/z:
calcd for C17H23N3 [M]+: 269.1892; found 269.1899.
l
nylpiperazin-1-amine and further stirring for 3 h were necessary
to reach full conversion of aldehyde. Yield: 119.0 mg (99%), color-
less solid. Rf (EtOAc/MeOH 30:1): 0.27. 1H NMR (300 MHz, DMSO-
d6): d (ppm) = 10.64 (br s, 2H, 2NH), 7.73 (s, 1H, CH@N), 7.26–7.21
(m, 3H, Ar-H), 7.18–7.14 (m, 1H, Ar-H), 7.01 (d, 3J = 8.1 Hz, 2H, Ar-
CH), 6.90 (d, 3J = 8.1 Hz, 1H, Ar-H), 6.81 (t, 3J = 7.2 Hz, 1H, Ar-H),
3.33–3.30 (m, 4H, 2CH2), 3.22–3.20 (m, 4H, 2CH2). 13C NMR
(75.5 MHz, DMSO-d6): d (ppm) = 155.4 (C@O), 150.6 (Cq), 137.4
(CH@N), 130.0 (Cq), 129.1 (Cq), 128.9 (2CHAr), 119.9 (CHAr),
119.1 (CHAr), 115.8 (2CHAr), 115.3 (Cq), 108.2 (CHAr), 104.9
(CHAr), 50.9 (2CH2), 47.8 (2CH2). Mp: 320–330 °C (dec.). HRMS
(EI+): m/z: calcd for C18H19ON5 [M]+: 321.1590; found 321.1606.
4.2.34. (E)-4-Phenyl-N-(pyridine-2-ylmethylen)piperazin-1-amine
(2e)
According to GP-1: 50.0 mg (282
perazin-1-amine, 30.2 mg (27.0
pyridinecarboxaldehyde, 1.0 mL toluene, stirring for 4.5 h.
Additional 3.0 mg (28.0 mol, 0.10 equiv) 2-pyridinecarboxalde-
l
mol, 2.00 equiv) 4-phenylpi-
l
L, 282 mol, 1.00 equiv) 2-
l
4.2.30. (E)-N-Pentyliden-4-phenylpiperazin-1-amine (2a)
According to GP-1: 50.0 mg (282
lmol, 1.00 equiv) 4-phenylpi-
l
perazin-1-amine, 24.3 mg (30.0 L, 282
l
lmol, 1.00 equiv) valer-
hyde and further stirring overnight were necessary to reach full
conversion of amine. Yield: 65.0 mg (87%), brown solid. 1H NMR
(300 MHz, CDCl3): d (ppm) = 8.38–8.36 (m, 1H, Ar-H), 7.71–7.69
(m, 1H, Ar-H), 7.51 (s, 1H, CH@N), 7.15–7.08 (m, 3H, Ar-H), 7.00–
6.97 (m, 1H, Ar-H), 6.81 (d, 3J = 7.8 Hz, 2H, Ar-H), 6.73 (t,
3J = 7.2 Hz, 1H, Ar-H), 3.23–3.20 (m, 8H, 4CH2). 13C NMR
aldehyde, 1.0 mL toluene, stirring overnight. Yield: 65.0 mg (94%),
orange oil. 1H NMR (300 MHz, CDCl3): d (ppm) = 7.31–7.25 (m,
2H, Ar-H), 7.05 (t, 3J = 5.4 Hz, 1H, CH@N), 6.96 (d, 3J = 7.8 Hz, 2H,
Ar-H), 6.88 (t, 3J = 7.2 Hz, 1H, Ar-H), 3.34 (t, 3J = 5.1 Hz, 4H, 2CH2),
3.12 (t, 3J = 5.1 Hz, 4H, 2CH2), 2.28 (q, 3J = 7.2 Hz, 2H, CH2), 1.55–
1.32 (m, 4H, 2CH2), 0.93 (t, 3J = 7.2 Hz, 3H, CH3). 13C NMR
(75.5 MHz, CDCl3): d (ppm) = 150.9 (CH@N), 143.5 (Cq), 129.1
(2CHAr), 120.1 (CHAr), 116.4 (2CHAr), 51.9 (2CH2), 48.8 (2CH2),
32.8 (CH2), 29.5 (CH2), 22.3 (CH2), 13.9 (CH3). HRMS (EI+): m/z:
calcd for C15H23N3 [M]+: 245.1892; found 245.1885.
(75.5 MHz, CDCl3):
d (ppm) = 155.2 (Cq), 150.9 (Cq), 149.0
(CHAr), 136.3 (CH@N), 135.9 (CHAr), 129.2 (2CHAr), 122.4
(CHAr), 120.3 (CHAr), 119.3 (CHAr), 116.6 (2CHAr), 50.7 (2CH2),
48.9 (2CH2). Mp: 117-119 °C.
4.2.35. (E)-4-Phenyl-N-(pyridine-3-ylmethylen)piperazin-1-amine
4.2.31. (E)-N-((E)-Hex-2-enyliden)-4-phenylpiperazin-1-amine
(2f)
(2b)
According to GP-1: 50.0 mg (282
perazin-1-amine, 30.2 mg (26.5
l
mol, 1.00 equiv) 4-phenylpi-
According to GP-1: 51.1 mg (289
perazin-1-amine, 28.3 mg (30.0 L, 289
hexen-1-al, 3.0 mL toluene, stirring for 3 h. Yield: 74.3 mg (>99%),
brown solid. 1H NMR (300 MHz, CDCl3):
(ppm) = 7.38 (d,
l
mol, 1.00 equiv) 4-phenylpi-
l
L, 282 mol, 1.00 equiv) 3-
l
l
lmol, 1.00 equiv) trans-2-
pyridinecarboxaldehyde, 1.0 mL toluene, stirring for 5 h. Yield:
75.0 mg (99%), red-brown solid. 1H NMR (300 MHz, CDCl3): d
(ppm) = 8.75 (d, 4J = 1.8 Hz, 1H, Ar-H), 8.51–8.49 (m, 1H, Ar-H),
8.02–7.98 (m, 1H, Ar-H), 7.56 (s, 1H, CH@N), 7.33–7.25 (m, 3H,
Ar-H), 6.99 (d, 3J = 8.1 Hz, 2H, Ar-H), 6.91 (t, 3J = 7.2 Hz, 1H, Ar-
d
3J = 8.7 Hz, 1H, CH@N), 7.31–7.27 (m, 2H, Ar-H), 6.98 (d,
3J = 7.8 Hz, 2H, Ar-H), 6.89 (t, 3J = 7.2 Hz, 1H, Ar-H), 6.28–6.20 (m,
1H, CH), 5.99–5.89 (m, 1H, CH), 3.36–3.33 (m, 4H, 2CH2), 3.22–
3.19 (m, 4H, 2CH2), 2.20–2.12 (m, 2H, CH2), 1.47 (m, 2H, CH2),
0.93 (t, 3J = 7.2 Hz, 3H, CH3). 13C NMR (75.5 MHz, CDCl3): d
(ppm) = 150.9 (Cq), 140.6 (CH), 138.4 (CH@N), 129.1 (2CHAr),
128.7 (CH), 120.1 (CHAr), 116.5 (2CHAr), 51.3 (2CH2), 48.8
H), 3.39 (s, 8H,
4 d
CH2). 13C NMR (75.5 MHz, CDCl3):
(ppm) = 150.8 (Cq), 149.0 (CHAr), 148.2 (CHAr), 132.3 (CH@N),
132.2 (CHAr), 132.0 (Cq), 129.2 (2CHAr), 123.5 (CHAr), 120.4
(CHAr), 116.6 (2CHAr), 50.8 (2CH2), 48.9 (2CH2). Mp: 111–113 °C

N. Mayer et al. / Bioorg. Med. Chem. 23 (2015) 2904–2916
2913
4.2.36. (E)-4-Phenyl-N-(pyridine-4-ylmethylen)piperazin-1-amine
(2g)
According to GP-1: 50.0 mg (282
perazin-1-amine, 30.2 mg (26.5
pyridinecarboxaldehyde, 1.0 mL toluene, stirring for 5 h. Yield:
75.0 mg (99%), orange solid. 1H NMR (300 MHz, CDCl3):
4.2.40. (E)-4-((4-(Pyridine-4-yl)piperazin-1-ylimino)methyl)-
benzene-1,3-diol (3a)
According to GP-1: 250 mg (1.40 mmol, 1.00 equiv) 4-(pyri-
dine-4-yl)piperazine-1-amine), 4.0 mL toluene, 190 mg
l
mol, 1.00 equiv) 4-phenylpi-
l
L, 282 mol, 1.00 equiv) 4-
l
(1.40 mmol, 1.00 equiv) 2,4-dihydroxybenzaldehyde. Stirring at
100 °C for 2 h. Trituration with 15 mL hot toluene yielded the pure
product. Yield: 310.0 mg (74%), brown solid. Rf (MeOH): 0.30. 1H
NMR (300 MHz, DMSO-d6): d (ppm) = 11.52 (br s, 1H, OH), 9.90
(br s, 1H, OH), 8.19 (br s, 2H, Ar-H), 7.97 (s, 1H, CH@N), 7.17 (d,
3J = 8.4 Hz, 1H, Ar-H), 6.88 (d, 3J = 6.0 Hz, 2H, Ar-H), 6.31 (dd,
3J = 8.4 Hz, 4J = 2.1 Hz, 1H, Ar-H), 6.24 (d, 4J = 2.1 Hz, 1H, Ar-H),
3.53–3.49 (m, 4H, 2CH2), 3.15–3.12 (m, 4H, 2CH2). 13C NMR
(75.5 MHz, DMSO-d6): d (ppm) = 159.1 (Cq-OH), 158.6 (Cq-OH),
154.1 (Cq), 149.8 (2CHAr), 142.7 (CH@N), 130.8 (CHAr), 111.4
(Cq), 108.6 (2CHAr), 107.0 (CHAr), 102.4 (CHAr), 50.7 (2CH2),
44.6 (2CH2). Mp: 270–273 °C (dec.). HRMS (EI+): m/z: calcd for
C16H18O2N4 [M]+: 298.1430; found 298.1447.
d
(ppm) = 8.55 (d, 3J = 6.0 Hz, 2H, Ar-H), 7.46 (d, 3J = 6.0 Hz, 2H, Ar-
H), 7.43 (s, 1H, CH@N), 7.33–7.27 (m, 2H, Ar-H), 6.98 (d,
3J = 7.8 Hz, 2H, Ar-H), 6.92 (t, 3J =7.2 Hz, 1H, Ar-H), 3.41–3.40 (m,
8H, 4 CH2). 13C NMR (75.5 MHz, CDCl3): d (ppm) = 150.8 (Cq),
149.8 (2CHAr), 143.6 (Cq), 131.6 (CH@N), 129.2 (2CHAr), 120.5
(CHAr), 120.1 (2CHAr), 116.7 (2CHAr), 50.6 (2CH2), 48.9 (2CH2).
Mp: 156–158 °C.
4.2.37. (E)-N-(Naphthalene-2-ylmethylen)-4-phenylpiperazin-
1-amine (2h)
According to GP-1: 40.0 mg (226
perazin-1-amine, 35.3 mg (226
l
mol, 2.00 equiv) 4-phenylpi-
l
mol, 1.00 equiv) 2-naph-
4.2.41. (E)-4-((4-(4-Fluorophenyl)piperazin-1-ylimino)methyl)-
benzene-1,3-diol (3b)
thaldehyde, 1.0 mL toluene, stirring overnight. Addition of 2.0 mg
(13.0 mol, 0.06 equiv) 4-phenylpiperazin-1-amine after 18 h to
l
According to GP-1: 150 mg (768
lmol, 1.00 equiv) 4-(4-fluo-
reach full conversion of the starting material after further stirring
for 6 h. Yield: 61.0 mg (82%), yellow solid. 1H NMR (300 MHz,
CDCl3): d (ppm) = 7.98–7.96 (m, 1H, Ar-H), 7.88 (s, 1H, CH@N),
7.84–7.80 (m, 4H, Ar-H), 7.48–7.46 (m, 2H, Ar-H), 7.34–7.29 (m,
2H, Ar-H), 7.02 (d, 3J = 8.1 Hz, 2H, Ar-H), 6.95–6.90 (m, 1H, Ar-H),
3.42 (s, 8H, 4CH2). 13C NMR (75.5 MHz, CDCl3): d (ppm) = 150.9
(Cq), 136.6 (CH@N), 133.8 (Cq), 133.5 (Cq), 133.4 (Cq), 129.2
(2CHAr), 128.3 (CHAr), 128.0 (CHAr), 127.8 (CHAr), 126.6 (CHAr),
126.2 (CHAr), 126.0 (CHAr), 123.1 (CHAr), 120.3 (CHAr), 116.6
(2CHAr), 51.2 (2CH2), 49.0 (2CH2). Mp: 195–198 °C (dec.). HRMS
(EI+): m/z: calcd for C21H21N3 [M]+: 315.1736; found 315.1738.
rophenyl)piperazine-1-amine, 2.0 mL toluene, 110 mg (768
lmol,
1.00 equiv) 2,4-dihydroxybenzaldehyde. Stirring at 100 °C for 2 h.
Recrystallization from 7 mL toluene yielded the pure product.
Yield: 67.8 mg (25%), brown solid. Rf (MeOH): 0.64. 1H NMR
(300 MHz, DMSO-d6): d (ppm) = 11.59 (br s, 1H, OH), 9.72 (br s,
1H, OH), 7.95 (s, 1H, CH@N), 7.17 (d, 3J = 8.4 Hz, 1H, Ar-H), 7.10–
6.99 (m, 4H, Ar-H), 6.31 (dd, 3J = 8.4 Hz, 4J = 2.4 Hz, 1H, Ar-H),
6.25 (d, 4J = 2.4 Hz, 1H, Ar-H), 3.26–3.25 (m, 4H, 2CH2), 3.18–3.16
(m, 4H, 2CH2). 13C NMR (75.5 MHz, DMSO-d6): d (ppm) = 159.0
(Cq-OH), 158.6 (Cq-OH), 157.7, 154.6 (1JC–F = 236.0 Hz, Cq-F),
147.4, 147.3 (4JC–F = 2.0 Hz, Cq), 142.3 (CH@N), 130.7 (CHAr),
117.7, 117.6 (3JC–F = 7.6 Hz, 2CHAr), 115.4, 115.1 (2JC–F = 21.9 Hz,
2CHAr), 111.4 (Cq), 107.0 (CHAr), 102.4 (CHAr), 51.1 (2CH2), 48.3
(2CH2). Mp: 176–178 °C.
4.2.38. (E)-4-Phenyl-N-(quinoline-2-ylmethylen)-piperazin-1-
amine (2i)
According to GP-1: 40.0 mg (226
perazin-1-amine, 35.5 mg (226 mol, 1.00 equiv) 2-quinolinecar-
boxaldehyde, 1.0 mL toluene, stirring for 5.5 h. Yield: 59.0 mg
lmol, 1.00 equiv) 4-phenylpi-
l
4.2.42. (E)-4-((4-m-Tolylpiperazin-1-ylimino)methyl)benzene-
1,3-diol (3c)
(83%), dark yellow solid. 1H NMR (300 MHz, CDCl3):
d
According to GP-1: 150 mg (731
lmol, 1.00 equiv) 4-m-
(ppm) = 8.09 (s, 2H, Ar-H), 8.05 (d, 3J = 8.4 Hz, 1H, Ar-H), 7.86 (s,
1H, CH@N), 7.78 (dd, 3J = 8.1 Hz, 4J = 0.6 Hz, 1H, Ar-H), 7.72–7.67
(m, 1H, Ar-H), 7.52–7.47 (m, 1H, Ar-H), 7.33–7.28 (m, 2H, Ar-H),
7.00 (d, 3J = 7.8 Hz, 2H, Ar-H), 6.92 (t, 3J = 7.2 Hz, 1H, Ar-H), 3.52–
3.49 (m, 4H, 2CH2), 3.43–3.39 (m, 4H, 2CH2). 13C NMR (75.5 MHz,
tolylpiperazine-1-amine, 2.0 mL toluene, 101 mg (731
l
mol,
1.00 equiv) 2,4-dihydroxybenzaldehyde. Stirring at 100 °C for 2 h.
Recrystallization from 2 mL toluene yielded the pure product.
Yield: 141.8 mg (58%), brown solid. Rf (MeOH): 0.65. 1H NMR
(300 MHz, DMSO- d6): d (ppm) = 11.60 (s, 1H, OH), 9.75 (br s, 1H,
OH), 7.95 (s, 1H, CH@N), 7.18–7.09 (m, 2H, Ar-H), 6.82–6.78 (m,
2H, Ar-H), 6.64 (d, 3J = 7.2 Hz, 1H, Ar-H), 6.31 (dd, 3J = 8.1,
4J = 2.1 Hz, 1H, Ar-H), 6.25 (d, 4J = 2.1 Hz, 1H, Ar-H), 3.31–3.28
(m, 4H, 2CH2), 3.17–3.15 (m, 4H, 2CH2), 2.26 (s, 3H, CH3). 13C
NMR (75.5 MHz, DMSO-d6): d (ppm) = 159.0 (Cq-OH), 158.6 (Cq-
OH), 150.5 (Cq), 142.3 (CH@N), 138.0 (Cq-CH3), 130.7 (CHAr),
128.7 (CHAr), 120.0 (CHAr), 116.4 (CHAr), 113.0 (CHAr), 111.4
(Cq), 107.0 (CHAr), 102.4 (CHAr), 51.1 (2CH2), 47.6 (2CH2), 21.3
(CH3). Mp: 135–139 °C. HRMS (EI+): m/z: calcd for C18H21O2N3
[M]+: 311.1654; found 311.1634.
CDCl3):
d (ppm) = 155.6 (Cq), 150.8 (Cq), 147.7 (Cq), 136.1
(CH@N), 135.7 (Cq), 129.6 (CHAr), 129.2 (2CHAr), 128.7 (CHAr),
127.7 (CHAr), 127.6 (CHAr), 126.2 (CHAr), 120.4 (CHAr), 117.5
(CHAr), 116.7 (2CHAr), 50.7 (2CH2), 48.9 (2CH2). Mp: 210–212 °C.
HRMS (EI+): m/z: calcd for C20H20N4 [M]+: 316.1688; found
316.1696.
4.2.39. (E)-N-(Naphthalene-1-methylen)-4-phenylpiperazin-1-
amine (2j)
According to GP-1: 50.9 mg (288
perazin-1-amine, 44.9 mg (39.0 L, 288
thaldehyde, 3.0 mL toluene, stirring for 4 h. Yield: 88.6 mg (98%),
brown solid. 1H NMR (300 MHz, CDCl3):
(ppm) = 8.60 (d,
l
mol, 1.00 equiv) 4-phenylpi-
l
l
mol, 1.00 equiv) 1-naph-
4.2.43. (E)-4-((4-Benzylpiperazin-1-ylimino)methyl)benzene-
1,3-diol (3d)
d
3J = 8.1 Hz, 1H, Ar-H), 8.33 (s, 1H, CH@N), 7.91–7.87 (m, 2H, Ar-
H), 7.83 (d, 3 J = 8.1 Hz, 1H, Ar-H), 7.59–7.48 (m, 3H, Ar-H), 7.36–
7.30 (m, 2H, Ar-H), 7.03 (d, 3J = 7.8 Hz, 2H, Ar-H), 6.94 (t,
3J = 7.2 Hz, 1H, Ar-H), 3.47 (s, 8H, 4CH2). 13C NMR (75.5 MHz,
CDCl3): d (ppm) = 150.9 (Cq), 135.7 (CH@N), 133.9 (Cq), 131.5
(Cq), 130.7 (Cq), 129.2 (2CHAr), 128.8 (CHAr), 128.7 (CHAr),
126.3 (CHAr), 125.7 (CHAr), 125.5 (CHAr), 125.3 (CHAr), 123.8
(CHAr), 120.3 (CHAr), 116.6 (2CHAr), 51.3 (2CH2), 49.0 (2CH2).
Mp: 118 °C.
According to GP-1: 150 mg (785
zylpiperazin-1-amine, 2.0 mL toluene, 108 mg (785
l
mol, 1.00 equiv) 4-ben-
lmol,
1.00 equiv) 2,4-dihydroxybenzaldehyde. Stirring at 100 °C for 2 h.
Final purification by recrystallization from 4.0 mL toluene yielded
the pure product. Yield: 134.0 mg (95%), beige solid. 1H NMR
(300 MHz, DMSO-d6): d (ppm) = 11.61 (br s, 1H, OH), 9.67 (br s,
1H, OH), 7.83 (s, 1H, CH), 7.33–7.25 (m, 5H, Ar-H), 7.12 (d,
3J = 8.4 Hz, 1H, Ar-H), 6.30 (dd, 3J = 8.4 Hz, 4J = 2.1 Hz, 1H, Ar-H),
6.23 (d, 4J = 2.1 Hz, 1H, Ar-H), 3.52 (s, 2H, CH2), 3.03 (s, 4H,

2914
N. Mayer et al. / Bioorg. Med. Chem. 23 (2015) 2904–2916
2CH2), 2.53 (s, 4H, 2CH2). 13C NMR (75.5 MHz, DMSO-d6): d
(ppm) = 158.8 (Cq-OH), 158.5 (Cq-OH), 141.3 (CH@N), 137.9 (Cq),
130.6 (CHAr), 128.7 (2CHAr), 128.1 (2CHAr), 126.9 (CHAr), 111.5
(Cq), 106.9 (CHAr), 102.3 (CHAr), 61.6 (CH2), 51.4 (2CH2), 51.1
(2CH2). Mp: 225–228 °C. HRMS (EI+): m/z: calcd for C18H21O2N3
[M]+: 311.1634; found 311.1656.
1.00 equiv) 2,4-dihydroxybenzaldehyde. Stirring at 100 °C for
2.5 h. Final purification by recrystallization from 2.0 mL toluene
yielded the pure product. Yield: 48.0 mg (57%), yellow crystals.
1H NMR (300 MHz, CDCl3): d (ppm) = 11.77 (s, 1H, OH), 9.66 (br
s, 1H, OH), 7.89 (s, 1H, CH@N), 7.34–7.27 (m, 4H, Ar-H), 7.23–
7.20 (m, 1H, Ar-H), 7.14 (d, 3J = 8.4 Hz; 1H, Ar-H), 6.29 (dd,
3J = 8.4 Hz, 4J = 2.4 Hz, 1H, Ar-H), 6.23 (d, 4J = 2.4 Hz, 1H, Ar-H)
3.74–3.70 (m, 2H, CH2), 2.67–2.59 (m, 3H, CH, CH2), 1.88–1.80
(m, 4H, 2CH2). 13C NMR (75.5 MHz, CDCl3): d (ppm) = 158.7 (Cq-
OH), 158.5 (Cq-OH), 145.5 (Cq), 141.1 (CH@N), 130.6 (CHAr),
128.3 (2CHAr), 126.6 (2CHAr), 126.1 (CHAr), 111.7 (Cq), 106.8
(CHAr), 102.3 (CHAr), 51.6 (2CH2), 41.0 (CH), 31.7 (2CH2). Mp:
139–142 °C. HRMS (EI+): m/z: calcd for C18H20O2N2 [M]+:
296.1525; found 296.1530.
4.2.44. (E)-4-((4-Phenethylpiperazin-1-ylimino)methyl)benzene-
1,3-diol (3e)
According to GP-1: 100 mg (488
4-phenethylpiperazin-1-amine, 2.0 mL
(439 mol, 1.00 equiv) 2,4-dihydroxybenzaldehyde. Stirring at
lmol, 1.00 equiv, 90% purity)
toluene, 60.7 mg
l
100 °C overnight. Final purification by recrystallization from
20.0 mL toluene yielded the pure product. Yield: 148.5 mg (94%),
orange-brown solid. 1H NMR (300 MHz, DMSO- d6):
d
(ppm) = 11.83 (br s, 1H, OH), 9.88 (br s, 1H, OH), 8.05 (s, 1H,
CH@N), 7.51–7.32 (m, 6H, Ar-H), 6.50 (dd, 3J = 8.4 Hz, 4J = 1.8 Hz,
1H, Ar-H), 6.43 (d, 4J = 1.8 Hz, 1H, Ar-H), 3.23 (br s, 2H, CH2),
2.98–2.93 (m, 2H, CH2), 2.83–2.70 (m, 8H, 4CH2). 13C NMR
(75.5 MHz, DMSO-d6): d (ppm) = 158.8 (Cq-OH), 158.5 (Cq-OH),
141.3 (CH@N), 140.2 (Cq), 130.6 (CHAr), 128.6 (2CHAr), 128.1
(2CHAr), 125.8 (CHAr), 111.5 (Cq), 106.9 (CHAr), 102.3 (CHAr),
59.2 (CH2), 51.6 (2CH2), 51.1 (2CH2), 32.8 (CH2). Mp: 211–214 °C.
HRMS (EI+): m/z: calcd for C19H23O2N3 [M]+: 325.1790; found
325.1799.
4.2.48. (E)-4-((4-benzylpiperidine-1-ylimino)methyl)benzene-
1,3-diol (3i)
According to GP-1: 86.0 mg (453
4-benzylpiperidine-1-amine (34), 2.0 mL toluene, 60.1 mg
(435 mol, 1.0 equiv) 2,4-dihydroxybenzaldehyde. Stirring at
lmol, 1.00 equiv, 96% purity)
l
100 °C overnight. Yield: 134.0 mg (95%), yellow-brown solid. 1H
NMR (300 MHz, MeOD): d (ppm) = 7.82 (s, 1H, CH@N), 7.29–7.24
(m, 2H, Ar-H), 7.19–7.16 (m, 3H, Ar-H), 7.04 (d, 3J = 8.4 Hz, 1H,
Ar-H), 6.30 (dd, 3J = 8.4 Hz, 4 J = 2.4 Hz, 1H, Ar-H), 6.25 (d,
4J = 2.4 Hz, 1H, Ar-H), 3.61–3.57 (m, 2H, CH2), 2.58 (d, 3J = 6.9 Hz,
2H, CH2), 2.54–2.46 (m, 2H, CH2), 1.79–1.75 (m, 2H, CH2), 1.71–
1.63 (m, 1H, CH), 1.50–1.41 (m, 2H, CH2). 13C NMR (75.5 MHz,
MeOD): d (ppm) = 160.6 (Cq-OH), 160.5 (Cq-OH), 144.5 (CH@N),
141.6 (Cq), 132.3 (CHAr), 130.2 (2CHAr), 129.3 (2CHAr), 127.0
(CHAr), 113.4 (Cq), 108.1 (CHAr), 103.7 (CHAr), 53.3 (2CH2), 43.7
(CH2), 38.8 (CH), 32.1 (2CH2). Mp: 138–141 °C. HRMS (EI+): m/z:
calcd for C19H22O2N2 [M]+: 310.1681; found 310.1683.
4.2.45. (E)-4-((4-Cyclohexylpiperazin-1-ylimino)methyl)benzene-
1,3-diol (3f)
According to GP-1: 150 mg (818
lmol, 1.00 equiv) 4-cyclo-
hexylpiperazine-1-amine, 2.0 mL toluene, 113 mg (818
lmol,
1.00 equiv) 2,4-dihydroxybenzaldehyde. Stirring at 100 °C for 2 h.
Trituration with 16 mL hot toluene yielded the pure product.
Yield: 110.0 mg (66%), brown solid. Rf (MeOH): 0.55. 1H NMR
(300 MHz, DMSO-d6): d (ppm) = 11.65 (s, 1H, OH), 9.69 (br s, 1H,
OH), 7.82 (s, 1H, CH@N), 7.11 (d, 3J = 8.4 Hz, 1H, Ar-H), 6.28 (dd,
3J = 8.4 Hz, 4J = 2.1 Hz, 1H, Ar-H), 6.21 (d, 4J = 2.1 Hz, 1H, Ar-H),
2.99 (br s, 4H, 2CH2), 2.65 (br s, 4H, 2CH2), 2.30–2.27 (m, 1H,
CH), 1.75–1.73 (m, 4H, 2CH2), 1.59–1.56 (m, 1H, CH), 1.19–1.05
4.2.49. (E)-4-((4-Phenyl-1,4-diazepan-1-ylimino)methyl)-
benzene-1,3-diol (3j)
According to GP-1: 31.7 mg (166
1,4-diazepane-1-amine (40), 1.0 mL toluene, 21.9 mg (166
l
mol, 1.00 equiv) 4-phenyl-
mol,
l
1.00 equiv) 2,4-dihydroxybenzaldehyde. Stirring at 100 °C over-
night. Yield: 50.0 mg (97%), brown-red oil. Rf (CH/EtOAc 3:1):
0.75. 1H NMR (300 MHz, DMSO-d6): d (ppm) = 11.39 (s, 1H, CH),
9.51 (br s, 1H, OH), 7.54 (s, 1H, OH), 7.18–7.12 (m, 2H, Ar-H),
7.06 (d, 3J = 8.4 Hz, 1 H, Ar-H), 6.75 (d, 3J = 8.4 Hz, 1 H, Ar-H),
6.58 (t, 3J = 7.2 Hz, 1H, Ar-H), 6.25 (dd, 3J = 8.4 Hz, 4J = 2.1 Hz, 1H,
Ar-H), 6.19 (d, 4J = 2.1 Hz, 1H, Ar-H), 3.63–3.62 (m, 2H, CH2),
3.52–3.50 (m, 2H, CH2), 3.43–3.37 (m, 4H, 2CH2), 1.99–1.95 (m,
2H, CH2). 13C NMR (75.5 MHz, DMSO-d6): d (ppm) = = 157.8 (Cq-
OH), 157.5 (Cq-OH), 147.1 (CH@N), 135.5 (Cq), 129.4 (CHAr),
129.2 (2CHAr), 115.5 (CHAr), 112.5 (Cq), 111.4 (2CHAr), 106.7
(CHAr), 102.3 (CHAr), 51.7 (CH2), 49.6 (CH2), 47.3 (CH2), 47.2
(CH2), 23.2 (CH2). HRMS (EI+): m/z: calcd for C18H21O2N3 [M]+:
311.1634; found 311.1616.
(m, 5H, 2CH2, CH). 13C NMR (75.5 MHz, DMSO-d6):
d
(ppm) = 158.8 (Cq-OH), 158.5 (Cq-OH), 141.1 (CH@N), 130.6
(CHAr), 111.5 (Cq), 106.9 (CHAr), 102.3 (CHAr), 62.2 (CH), 51.6
(2CH2), 47.4 (2CH2), 28.3 (2CH2), 25.8 (CH2), 25.2 (2CH2). Mp:
247–249 °C (dec.). HRMS (EI+): m/z: calcd for C17H25O2N3 [M]+:
303.1947; found 303.1942.
4.2.46. (E)-4-((4-Cyclopentylpiperazin-1-ylimino)methyl)-
benzene-1,3-diol (3g)
According to GP-1: 150 mg (887
lmol, 1.00 equiv) 4-
cyclopentylpiperazine-1-amine, 2.0 mL toluene, 123 mg (887
lmol,
1.00 equiv) 2,4-dihydroxybenzaldehyde. Stirring at 100 °C for 2 h.
Trituration with 16 mL hot toluene yielded the pure product. yield:
186.1 mg (76%), beige solid. Rf (MeOH): 0.57. 1H NMR (300 MHz,
DMSO-d6): d (ppm) = 11.63 (s, 1H, OH), 9.69 (br s, 1H, OH), 7.82 (s,
1H, CH@N), 7.12 (d, 3J = 8.4 Hz, 1H, Ar-H), 6.28 (dd, 3J = 8.4 Hz,
4J = 2.1 Hz, 1H, Ar-H), 6.22 (d, 4J = 2.1 Hz, 1H, Ar-H), 3.00 (br s, 4H,
2CH2), 2.58–2.49 (m, 4H, 2CH2), 2.46–2.44 (m, 1H, CH), 1.81–1.78
4.2.50. 4-((4-Phenylpiperazin-1-yl)methyl)benzene-1,2-diol (8)
A 25 mL Schlenk tube was flushed with argon and charged with
250 mg (1.81 mmol, 1.00 equiv) 3,4-dihydroxybenzaldehyde,
7.0 mL anhydrous DCE and 294 mg (0.28 mL, 1.81 mmol,
1.00 equiv) 1-phenylpiperazine. To the orange solution were added
538 mg (2.54 mmol, 1.40 equiv) sodium triacetoxyborhydride and
(m, 2H, CH2), 1.63–1.47 (m, 4H, 2CH2), 1.36–1.30 (m, 2H, CH2). 13
C
NMR (75.5 MHz, DMSO-d6): d (ppm) = 158.8 (Cq-OH), 158.5 (Cq-
OH), 141.2 (CH@N), 130.6 (CHAr), 111.5 (Cq), 106.9 (CHAr), 102.3
(CHAr), 66.3 (CH), 51.2 (2CH2), 50.6 (2CH2), 29.9 (2CH2), 23.6
(2CH2). Mp: 237–239 °C (dec.). HRMS (EI+): m/z: calcd for
100 lL (1.81 mmol, 1.00 equiv) acetic acid. The colorless suspen-
sion was stirred at rt overnight, during which the color of the sus-
pension turned bright yellow. The mixture was hydrolyzed by
addition of saturated NaHCO3 solution (10 mL) and concentrated
under reduced pressure. The residue was dissolved in EtOAc
(30 mL) and water (30 mL) and the layers were separated. The
aqueous layer was extracted with EtOAc (2 ꢁ 20 mL) and the com-
bined organic layers were washed with brine (25 mL), dried over
C
16H23O2N3 [M]+: 289.1790; found 289.1795.
4.2.47. (E)-4-((4-Phenylpiperidine-1-ylimino)methyl)benzene-
1,3-diol (3h)
According to GP-1: 50.0 mg (284
lmol, 1.00 equiv) 4-phenylpi-
peridine-1-amine (30), 1.0 mL toluene, 39.0 mg (284
lmol,

N. Mayer et al. / Bioorg. Med. Chem. 23 (2015) 2904–2916
2915
MgSO4 and concentrated under reduced pressure. Final purifica-
tion by silica gel filtration (DCM/MeOH 19:1, Rf = 0.38) yielded
the pure product. Yield: 135.9 mg (26%), brown solid. 1H NMR
(300 MHz, DMSO-d6): d (ppm) = 8.87 (br s, 2H, 2OH), 7.19 (t,
3J = 7.8 Hz, 2H, Ar-H), 6.90 (d, 3J = 8.1 Hz, 2H, Ar-H), 6.78–6.73
(m, 2H, Ar-H), 6.68–6.65 (m, 1H, Ar-H), 6.56–6.53 (m, 1H, Ar-H),
3.32 (s, 2H, CH2), 3.10 (br s, 4H, 2CH2), 2.47–2.46 (m, 4H, 2CH2).
13C NMR (75.5 MHz, DMSO-d6): d (ppm) = 150.9 (Cq), 144.9 (Cq-
OH), 144.1 (Cq-OH), 128.8 (2CHAr), 128.6 (Cq), 119.7 (CHAr),
118.6 (CHAr), 116.2 (CHAr), 115.2 (2CHAr), 115.0 (CHAr), 61.8
(CH2), 52.4 (2CH2), 48.1 (2CH2). Mp: 100–102 °C. HRMS (EI+): m/
z: calcd for C17H20O2N2 [M]+: 284.1525; found 284.1540.
2CH2), 3.17–3.13 (m, 4H, 2CH2). 13C NMR (75.5 MHz, DMSO-d6):
d (ppm) = 155.0 (C@O), 150.8 (Cq-OCH3), 148.3 (Cq-OCH3), 143.9
(Cq), 133.9 (Cq), 128.8 (2CHAr), 119.1 (CHAr), 115.7 (2CHAr),
112.0 (CHAr), 111.5 (CHAr), 105.3 (CHAr), 55.7 (OCH3), 55.2
(OCH3), 48.3 (2CH2), 43.5 (2CH2). Mp: 122 °C–125 °C.
4.2.53. 3,4-Difluorophenyl-4-phenylpiperazin-1-carboxylate
(14)
A 15 mL Schlenk tube was dried under vacuum, filled with
argon and charged with 500 mg (3.84 mmol, 1.00 equiv) 3,4-di-
fluorophenol, 2.0 mL absolute DCM and 624 mg (3.84 mmol,
1.00 equiv) 1,10-carbonyldiimidazole. The yellow mixture was stir-
red at rt for 1.5 h. To reach full conversion of the starting material
(GC–MS analysis) 605 mg (3.72 mmol, 0.97 equiv) 1,10-car-
bonyldiimidazol, dissolved in 1 mL DCM, were added and the mix-
ture was stirred at rt for further 20 h. The product solution was
used in the next reaction without work up or any purification step.
4.2.51. 3,4-Dimethoxy-N-(4-phenylpiperazin-1-yl)benzamide
(10)
A 25 mL Schlenk tube was charged with 100 mg (549
lmol,
1.00 equiv) 3,4-dimethoxybenzoic acid, 218 mg (140
lL,
1.83 mmol, 3.33 equiv) thionylchloride and one catalytic drop of
anhydrous DMF. The light yellow suspension was heated under
reflux for 3.5 h. The excess thionylchloride was removed under
high vacuum over a cooling trap to obtain a light yellow solid (acid
chloride). A 25 mL one-neck round bottom flask was charged with
623 mg (590
added to the product mixture of 3,4-difluorophenyl-1H-imidazol-
1-carboxylate. After 1 h stirring at rt once more 25.0 mg (20 L,
150 mol, 0.04 equiv) 1-phenylpiperazine were added to reach full
conversion (GC–MS analysis). The mixture was stirred at rt for 2 h
and washed with water (2 ꢁ 5 mL). The organic layer was dried
over MgSO4 and concentrated under reduced pressure. As the
obtained solid was not pure enough, even after silica gel filtration
(CH/EtOAc 5:1, Rf = 0.18), it was dissolved in DCM (5 mL) and
washed with 5 mL 2 M NaOH (5 mL). The organic layer was dried
over Na2SO4, concentrated under reduced pressure and dried under
high vacuum to yield the pure product. Yield: 246.0 mg (20%), light
lL, 3.84 mmol, 1.00 equiv) 1-phenylpiperazine were
l
l
117 mg (659 lmol, 1.20 equiv) 4-phenylpiperazin-1-amine and
2.0 mL 10% aqueous NaOH (2.0 mL). The solution was cooled to
0 °C and a solution of the acid chloride in 1 mL absolute DCM
was added. The mixture was stirred at rt for 30 min, during which
a colorless solid precipitated. The solid was collected by filtration,
washed with DCM and dried under vacuum. Final purification by
recrystallization from EtOH (25 mL) yielded the pure product.
Yield: 98.0 mg (53%), colorless solid. 1H NMR (300 MHz, CDCl3): d
(ppm) = 7.40 (br s, 1H, NH), 7.31–7.27 (m, 3H, Ar-H), 6.98–6.84
(m, 5H, Ar-H), 3.93–3.92 (m, 6H, 2OCH3), 3.40 (m, 4H, 2CH2),
3.12 (m, 4H, 2CH2). 13C NMR (75.5 MHz, CDCl3): d (ppm) = 165.2
(C@O), 151.9 (Cq-OCH3), 150.9 (Cq-OCH3), 149.1 (Cq), 129.2
(2CHAr), 126.2 (Cq), 120.3 (CHAr), 119.4 (CHAr), 116.5 (2CHAr),
110.8 (CHAr), 110.2 (CHAr), 56.0 (2CH2), 55.4 (2OCH3), 48.9 (2CH2).
yellow solid. Rf (DCM): 0.49. 1H NMR (300 MHz, CDCl3):
d
(ppm) = 7.34–7.29 (m, 2H, Ar-H), 7.15 (q, 3J = 9.0 Hz, Ar-H), 7.07–
6.86 (m, 5H, Ar-H), 3.82–3.75 (m, 4H, 2CH2), 3.25 (t, 3J = 5.4 Hz,
4H, 2CH2). 13C NMR (75.5 MHz, CDCl3): d (ppm) = 153.0 (C@O),
2
151.7, 151.5, 148.4, 148.2 (1JC–F = 249.5 Hz, JC–F = 13.9 Hz, Cq-F),
2
149.7, 149.6, 146.5, 146.3 (1JC–F = 245.7 Hz, JC–F = 12.5 Hz, Cq-F),
4
147.0, 146.9, 146.8, (3JC–F = 8.8 Hz, JC–F = 3.2 Hz, Cq), 129.3
(2CHAr), 120.9 (CHAr), 117.7, 117.6, 117.5 (CHAr), 117.2 (Cq),
116.9 (2CHAr), 111.9, 111.6 (2JC–F = 19.9 Hz, CHAr), 49.6 (2CH2),
43.9 (2CH2). Mp: 94–96 °C. HRMS (EI+): m/z: calcd for
4.2.52. N-(3,4-Dimethoxyphenyl)-4-phenylpiperazin-1-
carboxamide (12)
A 25 mL Schlenk tube was dried under vacuum, filled with
argon and charged with 500 mg (3.26 mmol, 1.00 equiv) 3,4-
dimethoxyaniline and 6.1 mL absolute EtOH. 1.07 g (4.90 mmol,
1.50 equiv) di-tert-butyl dicarbonate were added slowly and the
suspension was stirred at rt for 2.5 h. The mixture was transferred
to a one-neck round bottom flask and concentrated under reduced
pressure. Drying under high vacuum overnight yielded the product
which was used in the next reaction without further purification. A
15 mL Schlenk tube was dried under vacuum, filled with argon and
C
17H16O2N2F2 [M]+: 318.1180; found 318.1182.
4.3. Triglyceride hydrolase activity assay
For the determination of TG hydrolase activity cell lysates of
COS-7 cells overexpressing recombinant murine ATGL were used.
Therefore, monkey embryonic kidney cells (Cos-7; ATCC CRL-
1651) were cultivated in Dulbeccos modified eagles medium
(DMEM, GIBCO, Invitrogen Corp., Carlsbad, CA), containing 10%
fetal calf serum (FCS, Sigma–Aldrich) and antibiotics (100 IU/mL
charged with 176 mg (166
phenylpiperazine and 2.0 mL absolute THF. The yellow solution
was cooled to 0 °C and 76.0 mg (474 L, 1.18 mmol, 1.20 equiv)
n-butyllithium were added. The mixture was warmed to rt and a
solution of 250 mg (987 mol, 1.00 equiv) of previously prepared
lL, 1.09 mmol, 1.10 equiv) 1-
penicillin and 100
(37 °C, 5% CO2, 95% humidified atmosphere). Cells were transfected
with 1 g DNA complexed to Metafectene (Biontex GmbH, Munich,
lg/mL streptomycin) at standard conditions
l
l
l
Germany) in serum free DMEM. After 4 h the medium was replaced
by DMEM supplemented with 10% FCS. For the preparation of cell
lysates, cells were washed with 1ꢁ phosphate buffered saline, col-
lected using a cell scraper, and disrupted in buffer A (0.25 M
tert-butyl-3,4-dimethoxyphenylcarbamate in 2.0 mL absolute THF
was added dropwise. The greenbrown solution was heated under
reflux for 5 h. The mixture was hydrolyzed with 5 mL 5% aqueous
HCl (5 mL). The aqueous layer was extracted with DCM (3 ꢁ 5 mL)
and the combined organic layers were washed with 5 mL saturated
aqueous NaHCO3 (5 mL), water (5 mL) and brine (5 mL). The
organic layer was dried over Na2SO4 and concentrated under
reduced pressure. Final purification by column chromatography
(CH/EtOAc, size: 18.5 ꢁ 2.5 cm, 30 g SiO2) yielded the pure product.
Yield: 81.0 mg (25%), yellow-orange solid. Rf (CH/EtOAc 1:1): 0.19.
1H NMR (300 MHz, DMSO-d6): d (ppm) = 8.44 (br, 1H, NH), 7.26–
7.17 (m, 3H, Ar-H), 7.00–6.97 (m, 3H, Ar-H), 6.84–6.79 (m, 2H,
Ar-H), 3.71 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 3.59–3.56 (m, 4H,
sucrose, 1 mM EDTA, 1 mM dithiothreitol, 20
lg/mL leupeptine,
2
l
g/mL antipain, g/mL pepstatin, pH 7.0) by sonication
1 l
(Virsonic 475, Virtis, Gardiner, NJ). Nuclei and unbroken cells were
removed by centrifugation (1000ꢁg, 4 °C, for 10 min). Protein con-
centration of the cell lysates were determined by Bio-Rad protein
assay according to the manufacturer’s protocol (Bio-Rad 785, Bio-
Rad Laboratories, Munich, Germany), using BSA as standard.
20
The triolein (TO) substrate was prepared as described.
brief, 330
emulsified with 45
In
l
M triolein (40,000 cpm/nmol) (GE Healthcare) was
M phosphatidylcholine/phosphatidylinositol
l

2916
N. Mayer et al. / Bioorg. Med. Chem. 23 (2015) 2904–2916
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acid free bovine serum albumin (BSA, Sigma). 100
tein) cell lysate or 2 l purified recombinant LPL (Sigma–Aldrich)
were incubated with 100 L TO substrate in the presence or in
lL (20 lg pro-
l
l
4. Fredrikson, G.; Stralfors, P.; Nilsson, N. O.; Belfrage, P. J. Biol. Chem. 1981, 256,
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This work was supported by the doctoral program DK Molecular
Enzymology (grant no. W901-B05, Ro. Zi., R. B.), Translational
Program (grant no. TRP4, Ro. Zi.), and the Wittgenstein Award
2007 (grant no. Z136, Ru. Ze.) funded by the Austrian
Science Fund (FWF), and GOLD, Genomics of Lipid-Associated
Disorders (Ru. Ze.), and PLACEBO, Platform for Chemical
Biology in Austria (R. B.), as part of the Austrian Genome Project
GEN-AU funded by the Forschungsförderungsgesellschaft (FFG)
und Bundesministerium für Wissenschaft und Forschung.
Furthermore, we thank P. Jacobsen and H. Tornqvist for the provi-
sion of hit compounds.
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Supplementary data
21. Recently, we have been reported that long chain acyl-CoA inhibits ATGL: Nagy,
H. M.; Paar, M.; Heier, C.; Moustafa, T.; Hofer, P.; Haemmerle, G.; Lass, A.;
Zechner, R.; Oberer, M.; Zimmermann, R. Biochim. Biophys. Acta 2014, 1841,
588.
22. A peptide based inhibitor of ATGL aciting in a non-competitive manner has
been described: Cerk, I. K.; Salzburger, B.; Boeszoermenyi, A.; Heier, C.; Pillip,
C.; Romauch, M.; Schweiger, M.; Cornaciu, I.; Lass, A.; Zimmermann, R.;
Zechner, R.; Oberer, M. J. Biol. Chem. 2014, 289, 32559.
Supplementary data (selectivity tests of compounds 1a and 1h
and toxicity test of 1h) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmc.2015.02.
051.
References and notes
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1. Lass, A.; Zimmermann, R.; Oberer, M.; Zechner, R. Prog. Lipid. Res. 2011, 50, 14.